TIGIT- and light-based chimeric proteins

ABSTRACT

The present invention relates, inter alia, to compositions and methods, including TIGIT- and/or LIGHT-based chimeric proteins that find use in the treatment of disease, such as cancer and an inflammatory disease.

PRIORITY

This application is a continuation of U.S. application Ser. No.16/484,850, filed Aug. 9, 2019. U.S. application Ser. No. 16/484,850 isthe 371 National Stage Entry of PCT/US18/20037, filed Feb. 27, 2018,which claims the benefit of, and priority to, U.S. ProvisionalApplication No. 62/464,002, filed Feb. 27, 2017, the contents of all ofwhich are hereby incorporated by reference in their entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

This application contains a sequence listing. It has been submittedelectronically via EFS-Web as an ASCII text file entitled“SHK-001US1C1_SequenceListing_ST25”. The sequence listing is 141,715bytes in size, and was created on Apr. 16, 2020. The sequence listing ishereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates to, inter alia, compositions and methods,including chimeric proteins that find use in the treatment of disease,such as immunotherapies for cancer and autoimmunity.

BACKGROUND

The immune system is central to the body's response to foreign entitiesthat can cause disease. However, many cancers have developed mechanismsto avoid the immune system by, for instance, delivering or propagatingimmune inhibitory signals. Thus, there remains a need to developtherapeutics that are endowed with multiple functionalities—forinstance, reversing immune inhibitory signal and stimulating ananti-cancer immune response.

SUMMARY

Accordingly, in various aspects, the present invention provides forcompositions and methods that are useful for cancer immunotherapy. Forinstance, the present invention, in part, relates to specific chimericproteins that reverse or suppresses immune inhibitory signals whileproviding immune activating or co-stimulatory signals. Importantly,inter alia, the present invention provides for improved chimericproteins that can maintain a stable and producible multimeric statebased on, without wishing to be bound by theory, stabilization in alinker region including one or more disulfide bonds. Accordingly, thepresent compositions and methods overcome various deficiencies inproducing bi-specific agents.

In some aspects, the chimeric protein is of a general structure of: Nterminus-(a)-(b)-(c)-C terminus, where (a) is a first domain comprisingan extracellular domain of a Type I transmembrane protein, (b) is alinker comprising at least one cysteine residue capable of forming adisulfide bond (including without limitation, hinge-CH2-CH3 Fc domain isderived from human IgG4), and (c) is a second domain comprising anextracellular domain of Type II transmembrane protein, where the linkerconnects the first domain and the second domain and optionally comprisesone or more joining linkers as described herein.

For instance, in embodiments, the extracellular domain of a Type Itransmembrane protein is from TIGIT (VSIG9, VSTM3).

In embodiments, the chimeric protein is of a general structure of: Nterminus-(a)-(b)-(c)-C terminus, where (a) is a first domain comprisingan extracellular domain of a Type I transmembrane protein, thetransmembrane protein being TIGIT, (b) is a linker comprising at leastone cysteine residue capable of forming a disulfide bond (includingwithout limitation, hinge-CH2-CH3 Fc domain is derived from human IgG4),and (c) is a second domain comprising an extracellular domain of Type IItransmembrane protein, the transmembrane protein being selected from4-1BBL (TNFSF9), GITRL (TNFSF18), TL1A (TNFSF15), and LIGHT (TNFSF14),where the linker connects the first domain and the second domain andoptionally comprises one or more joining linkers as described herein.

For instance, in embodiments, the extracellular domain of a Type IItransmembrane protein is from LIGHT.

In embodiments, the chimeric protein is of a general structure of: Nterminus-(a)-(b)-(c)-C terminus, where (a) is a first domain comprisingan extracellular domain of a Type I transmembrane protein, thetransmembrane protein being selected from PD-1, CD172a(SIRPα), andTIGIT, (b) is a linker comprising at least one cysteine residue capableof forming a disulfide bond (including without limitation, hinge-CH2-CH3Fc domain is derived from human IgG4), and (c) is a second domaincomprising an extracellular domain of Type II transmembrane protein, thetransmembrane protein being LIGHT, where the linker connects the firstdomain and the second domain and optionally comprises one or morejoining linkers as described herein.

In some aspects, there is provided a method for treating cancer or aninflammatory disease comprising administering an effective amount of apharmaceutical composition of the afore-mentioned chimeric proteins. Inembodiments, the subject's T cells are activated when bound by thesecond domain of the chimeric protein and (a) one or more tumor cellsare prevented from transmitting an immunosuppressive signal when boundby the first domain of the chimeric protein, (b) a quantifiable cytokineresponse in the peripheral blood of the subject is achieved, and/or (c)tumor growth is reduced in the subject in need thereof as compared to asubject treated with antibodies directed to the Type I or Type IIprotein, or their respective ligands or receptors. In embodiments, themethod stimulates signaling of one or more of LIGHT, 4-1BBL, GITRL, andTL1A and activates antigen-presenting cells. In embodiments, the methodreduces the amount or activity of regulatory T cells (Tregs) as comparedto untreated subjects or subjects treated with antibodies directed tothe Type I or Type II protein, or their respective ligands or receptors.In embodiments, the method increases priming of effector T cells indraining lymph nodes of the subject as compared to untreated subjects orsubjects treated with antibodies directed to the Type I or Type IIprotein, or their respective ligands or receptors. In embodiments, themethod causes an overall decrease in immunosuppressive cells and a shifttoward a more inflammatory tumor environment as compared to untreatedsubjects or subjects treated with antibodies directed to the Type I orType II protein, or their respective ligands or receptors.

Any aspect or embodiment described herein can be combined with any otheraspect or embodiment as disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A to FIG. 1D show schematic illustrations of how a Type I and TypeII membrane protein (FIG. 1A and FIG. 1C) may be engineered withtransmembrane and intracellular domains removed and adjoined using alinker sequence (FIG. 1B) to generate a single chimeric protein whereinthe extracellular domains of the Type I and Type II membrane proteinseach face outward in a single chimeric protein. FIG. 1B depicts thelinkage of a Type I and Type II membrane protein by removal of thetransmembrane and intracellular domains of each protein, and where theliberated extracellular domains (ECD) from each protein have beenadjoined by a linker sequence. The ECD in this depiction may include theentire amino acid sequence of a candidate Type I or Type II proteinwhich is typically localized outside the cell membrane, or any portionthereof which retains binding to the intended receptor or ligand. FIG.1D depicts adjoined extracellular domains in a linear construct whereinthe extracellular domain of the Type I membrane protein faces the “left”side of the construct and the extracellular domain of the Type IImembrane protein faces the “right” side of the construct.

FIG. 2A, using an PD-1-Fc-OX40L chimeric protein as an example, showsthat tumor cells may express PD-L1 on the cell surface, which can bindto PD-1 expressed by a T cell (FIG. 2B). This interaction suppressesactivation of T cells. A chimeric protein comprising the extracellulardomain of PD-1, adjoined to the extracellular domain of OX40L may bindto PD-L1 on the surface of a tumor cell, preventing binding to PD-1 onthe surface of a T cell (FIG. 2C). The chimeric protein may then“dangle” from the surface of the tumor cell, and the OX40L portion ofthe chimeric protein may then bind to OX40 expressed on the surface ofthe T cell. This would result in replacement of an inhibitory PD-L1signal with a co-stimulatory OX40L signal to enhance the anti-tumoractivity of T cells. FIG. 2D shows a synapse that has formed by achimeric protein between a tumor cell and a T cell. FIG. 2A to FIG. 2Cillustrate the mechanisms through which a PD-1-Fc-OX40L chimeric proteinbinds to its target molecules to creates a synapse between cells; thus,PD-1-Fc-OX40L is illustrative of the mechanism through which chimericproteins of the present invention operate.

FIG. 3A shows Western blots of murine TIGIT-Fc-OX40L chimeric proteinsrun on SDS-PAGE under reducing conditions, following treatment with N+Odeglycosylating enzyme, and/or after boiling.

FIG. 3B shows Western blots of human TIGIT-Fc-OX40L chimeric proteinsrun on SDS-PAGE under reducing conditions, following treatment with N+Odeglycosylating enzyme, and/or after boiling. In both cases, specificdetection of each domain is indicated by the anti-TIGIT, anti-Fc andanti-OX40L blots.

FIG. 4A are graphs showing ELISAs of murine TIGIT-Fc-OX40L chimericproteins performed under the following conditions: Heavy+Light Chaincaptured and detected with Fc-HRP (top left), CD155/PVR-His captured anddetected with IgG (top right), OX40-His captured and detected with anantibody directed to mOX40L (bottom left), and OX40-Fc captured anddetected with a recombinant CD155 (bottom right). FIG. 4B are graphsshowing ELISAs of human TIGIT-Fc-OX40L chimeric proteins performed underthe following conditions: Heavy+Light Chain captured and detected withFc-HRP (top left), CD155/PVR-His captured and detected with IgG (topright), OX40-His captured and detected with an antibody directed tomOX40L (bottom left), and OX40-Fc captured and detected with arecombinant CD155, CD112 or CD113 protein (bottom right).

FIG. 5A shows cell lines generated to overexpress human PVR (CHOK1/PVR),which could be used to detect binding via a TIGIT containing construct(in FIG. 5A, unstained and isotype are overlapping).

FIG. 5B shows cell lines generated to overexpress Nectin-2,(CHOK1/Nectin2), which could be used to detect binding via a TIGITcontaining construct.

FIG. 5C shows cell lines generated to overexpress Nectin-3(CHOK1/Nectin3), which could be used to detect binding via a TIGITcontaining construct (in FIG. 5C, unstained, isotype, and detection Abare overlapping).

FIG. 6 are graphs showing binding of mouse TIGIT-Fc-OX40L to CHO-K1cells expressing mouse PVR (top left), to parental CHO-K1 cells lackingPVR (bottom left) or to CHO-K1 cells expressing mouse OX40 (right).

FIG. 7 is a table of results showing the identified binding partners ofhuman TIGIT-Fc-OX40L from a microarray containing approximately 6,000human membrane proteins. In each case, the expected binding partners foreach candidate molecule were identified by the screen. There was noevidence of non-specific binding to other human proteins, and binding toGalectin-1 is seen in the screen for all Fc-containing fusion proteins.

FIG. 8A shows Western blots of murine mCD172a(SIRPα)-Fc-LIGHT chimericproteins run on SDS-PAGE under reducing conditions, following treatmentwith N+O deglycosylating enzyme, and/or after boiling.

FIG. 8B shows Western blots of human CD172a(SIRPα)-Fc-LIGHT chimericproteins run on SDS-PAGE under reducing conditions, following treatmentwith N+O deglycosylating enzyme, and/or after boiling.

FIG. 9A are graphs showing ELISAs of murine mCD172a(SIRPα)-Fc-LIGHTchimeric proteins performed under the following conditions: Heavy+LightChain captured and detected with Fc-HRP (top left), CD47-His capturedand detected with IgG (top right), mLTBR-His captured and detected withan antibody directed to mLIGHT (bottom left), and LTBR His+GST capturedand detected with an antibody directed to SIRPα (bottom right). FIG. 9Bare graphs showing ELISAs of human CD172a(SIRPα)-Fc-LIGHT chimericproteins performed under the following conditions: Heavy+Light Chaincaptured and detected with Fc-HRP (top left), CD47-His captured anddetected with IgG (top right), human LTBR-His captured and detected withan antibody directed to human LIGHT (bottom left), and LTBR His+GSTcaptured and detected with an antibody directed to SIRPα (bottom right).

FIG. 10A are graphs showing binding of mouse CD172a(SIRPα)-Fc-LIGHT toCHO-K1 cells expressing mouse CD47 (left) or to CHO-K1 cells expressingmouse LTbR (right). FIG. 10B are graphs showing binding of humanCD172a(SIRPα)-Fc-LIGHT to CHO-K1 cells expressing mouse CD47 (DetectionAb Only Control peak is far left, remainder of peak distribute left torighty by increasing concentration (i.e. 250 is far right)).

FIG. 11A are graphs showing binding of human CD172a(SIRPα)-Fc-LIGHT andCD172a(SIRPα)-Fc-CD40L to cynomolgus macaque red blood cells incomparison to CD47 specific antibodies (clone CC2C6 or CC900002), topleft. Lysis of cynomolgus macaque red blood cells following eachtreatment is shown over a titration curve in the bottom left and anexample plate is shown on right. Triton-X was used as a positive controlto cause lysis of cynomolgus macaque red blood cells (for reference, inthe top panel, at X-axis point 2 nM, curves top to bottom areanti-CD47/IgG-APC, anti-CD47-FITC, SIRPα-Fc-LIGHT, and SIRPα-Fc-CD40Lwhile in the bottom panel at X-axis point 2, curves top to bottom areTriton-X100, anti-CD47 (CC2C6) and anti-CD47 (CC9002), SIRPα-Fc-LIGHT,and SIRPα-Fc-CD40L all overlay). FIG. 11B are graphs showing binding ofhuman CD172a(SIRPα)-Fc-LIGHT and CD172a(SIRPα)-Fc-CD40L to human redblood cells in comparison to CD47 specific antibodies (clone CC2C6 orCC900002), from each of 3 different human blood donors. FIG. 11C, lysisof human red blood cells following each treatment is shown over atitration curve in the left and an example plate is shown on right.Triton-X was used as a positive control to cause lysis of cynomolgusmacaque red blood cells. Data are shown for 3 human red blood celldonors.

FIG. 12A shows Western blots of murine PD-1-Fc-LIGHT chimeric proteinsrun on SDS-PAGE under reducing conditions, following treatment with N+Odeglycosylating enzyme, and/or after boiling. FIG. 12B shows Westernblots of human PD-1-Fc-LIGHT chimeric proteins run on SDS-PAGE underreducing conditions, following treatment with N+O deglycosylatingenzyme, and/or after boiling.

FIG. 13A are graphs showing ELISAs of murine PD-1-Fc-LIGHT chimericproteins performed under the following conditions: Heavy+Light Chaincaptured and detected with Fc-HRP (left), mLTBR-His captured anddetected with an antibody directed to mLIGHT (middle), and mPD-L1captured and detected with an antibody directed to mLIGHT (right). FIG.13B are graphs showing ELISAs of human PD-1-Fc-LIGHT chimeric proteinsperformed under the following conditions: Heavy+Light Chain captured anddetected with Fc-HRP (left), hLTBR-Fc His captured and detected withbiotinylated hLIGHT (middle), and hPDL1-Fc captured and detected withhLTBR-His/6×His-HRP (right).

FIG. 14A are graphs showing binding of mouse PD-1-Fc-LIGHT to CHO-K1cells expressing mouse PD-L1 (left) or to CHO-K1 cells expressing mouseLTbR (right). FIG. 14B are graphs showing binding of human PD-1-Fc-LIGHTto CHO-K1 cells expressing human PD-L1.

FIG. 15A shows Western blots of murine TIGIT-Fc-LIGHT chimeric proteinsrun on SDS-PAGE under reducing conditions, following treatment with N+Odeglycosylating enzyme, and/or after boiling. FIG. 15B shows Westernblots of human TIGIT-Fc-LIGHT chimeric proteins run on SDS-PAGE underreducing conditions, following treatment with N+O deglycosylatingenzyme, and/or after boiling.

FIG. 16A are graphs showing ELISAs of murine TIGIT-Fc-LIGHT chimericproteins performed under the following conditions: Heavy+Light Chaincaptured and detected with Fc-HRP (left), CD155/PVR captured anddetected with Fc-HRP (middle), and mLTBR-His captured and detected withan antibody directed to mLIGHT (right). FIG. 16B are graphs showingELISAs of human TIGIT-Fc-LIGHT chimeric proteins performed under thefollowing conditions: Heavy+Light Chain captured and detected withFc-HRP (left), CD155-His captured and detected IgG (middle), andhCD155-Fc captured and detected with hLTBR-His/6×His-HRP (right).

FIG. 17 are graphs showing binding of mouse TIGIT-Fc-LIGHT to CHO-K1cells expressing mouse PVR (left) or to CHO-K1 cells expressing mouseLTbR (right).

FIG. 18A are graphs showing binding affinity measurements collected bybiolayer interferometry using an Octet system demonstrating binding ofhuman TIGIT-Fc-LIGHT, CD172a(SIRPα)-Fc-LIGHT and PD-1-Fc-LIGHT to humanLTbR (all panels, top to bottom: 30, 10, 3.3, 0). FIG. 18B are bindingaffinity measurements collected by biolayer interferometry using anOctet system demonstrating binding of human PD-1-Fc-LIGHT to recombinanthuman PD-L1 and PD-L2 across a range of concentrations (top to bottom:500 nM ARC×PD-L1, 500 nM ARC×PD-L2, 166 nM ARC×PD-L1, 166 nM ARC×PD-L2,56 nM ARC×PD-L1, 56 nM ARC×PD-L2, No ARC).

FIG. 19A are graphs showing binding affinity measurements collected bybiolayer interferometry using an Octet system demonstrating binding ofhuman TIGIT-Fc-OX40L and TIGIT-Fc-LIGHT to recombinant human CD155/PVRas compared to a one-sided TIGIT-Fc fusion protein control (top tobottom: TIGIT-Fc-LIGHT, TIGIT-Fc-Ox40L, TIGIT-Fc). FIG. 19B are bindingaffinity measurements collected by biolayer interferometry using anOctet system demonstrating binding of human CD172a(SIRPα)-Fc-LIGHT torecombinant human CD47 as compared to a single-sided CD172a(SIRPα)-Fccontrol, or one of the two CD47 specific antibody controls (top tobottom: SIRPα-Fc-LIGHT, anti-CD47, anti-CD47 CC2C6, and SIRP-Fc). FIG.19C are binding affinity measurements collected by biolayerinterferometry using an Octet system demonstrating binding of humanPD-1-Fc-LIGHT to recombinant human PD-L1 as compared to a single-sidedPD-1-Fc control or an anti-PD-L1 control (Atezolizumab) (top to bottom:PD-1-Fc-LIGHT, anti-PDL1, and PD-1-Fc). FIG. 19D are binding affinitymeasurements collected by biolayer interferometry using an Octet systemdemonstrating binding of human CD172a(SIRPα)-Fc-LIGHT, TIGIT-Fc-LIGHT orPD-1-Fc-LIGHT to recombinant human LTbR as compared to a single-sidedLIGHT-Fc fusion protein control or an anti-LTbR antibody (top to bottom:TIGIT-Fc-LIGHT, Sirp1a-Fc-LIGHT, anti-LTbR, PD-1-Fc-LIGHT, andLIGHT-Fc).

FIG. 20A and FIG. 20B show superantigen cytokine release assays whichdemonstrate the effects of the various antibodies, mouse TIGIT-Fc-OX40L,mouse CD172a(SIRPα)-Fc-LIGHT, mouse TIGIT-Fc-LIGHT, and mousePD-1-Fc-LIGHT chimeric proteins on mouse peripheral blood leukocytesactivated by staphylococcus enterotoxin B (SEB). FIG. 20A assays IL2secretion and FIG. 20B assays TNFα secretion. For FIG. 20A and FIG. 20B,the order of conditions for each concentration of SEB mirrors from leftto right, the conditions listed in the legends from top to bottom (e.g.α-PD-1 (RMP1-14) is the third from the top in the legends and istherefore third from the left in the graphs, PD-1-Fc-LIGHT (10 nM) isthe third from the bottom in the legends and is therefore third from theright in the graphs, and so forth). FIG. 20C shows a compilation of thedata across multiple superantigen (SEB) concentrations (again, the orderof conditions for each concentration of SEB mirrors from left to right,the conditions listed in the legends from top to bottom).

FIG. 21A to FIG. 21C show superantigen cytokine release assays whichdemonstrate the effects of the various antibodies, human TIGIT-Fc-LIGHT(FIG. 21A), human TIGIT-Fc-OX40L (FIG. 21B), human PD-1-Fc-LIGHT, andhuman CD172a(SIRPα)-Fc-LIGHT (FIG. 21C) chimeric proteins on humanperipheral blood leukocytes activated by staphylococcus enterotoxin B(SEB).

FIG. 22A and FIG. 22B show the results from in vivo tumor studiesdemonstrating the anti-tumor efficacy of mTIGIT-Fc-OX40L,mCD172a(SIRPα)-Fc-LIGHT, mTIGIT-Fc-LIGHT, and mPD-1-Fc-LIGHT chimericproteins in comparison with monoclonal antibodies to TIGIT, CD47, OX40,PD-1 or a combination of TIGIT and OX40. A CT26 tumor was implanted intoBalb/c mice prior to treatment with the indicated regimens. FIG. 22Ashows the evolution of tumor size over forty days after tumorinoculation for each group. FIG. 22B shows the overall survivalpercentage of mice through forty days after tumor inoculation and thenumber of complete tumor rejectors are indicate in the embedded table.In FIG. 22A and FIG. 22B, the treatment conditions are identified byletters.

FIG. 23 shows, without wishing to be bound by theory, four potentialconfigurations of PD-1-Fc-OX40L chimeric proteins.

FIG. 24 shows Western blots of PD-1-Fc-OX40L chimeric proteins run onSDS-PAGE under a non-reducing condition, a reducing condition, and areducing condition and following treatment with Peptide-N-Glycosidase F(PNGaseF).

FIG. 25 shows a chromatograph for PD-1-Fc-OX40L chimeric proteins run onSize Exclusion Chromatography (SEC).

FIG. 26 shows SDS-PAGE and native (non-SDS) PAGE gels for PD-1-Fc-OX40Lchimeric proteins run under a non-reducing condition (“−”) or a reducingcondition (“+”).

FIG. 27 shows a native (non-SDS) PAGE gel for PD-1-No Fc-OX40L chimericproteins which lack a Fc domain.

FIG. 28 shows, without wishing to be bound by theory, a model for how ahexamer and concatemers form from chimeric proteins of the presentinvention.

FIG. 29A to FIG. 29Q show characterization of PD-1-Fc-OX40L chimericproteins with different joining linker sequences by Western blotanalysis. Sequences of the different joining linkers are provided belowin the Examples section. Specifically, each individual domain of thefusion construct was probed using an α-PD-1, α-Fc, or α-OX40L antibody.In each figure, untreated samples of the PD-1-Fc-OX40L chimeric protein,e.g. control, were loaded into lane 1 in all the blots (no3-mercaptoethanol or PNGase). Samples in lane 2 were treated with thereducing agent, 3-mercaptoethanol, while samples in lane 3 were treatedwith PNGase.

FIG. 30 shows characterization of PD-1-Fc-OX40L chimeric proteins withdifferent joining linker sequences by ELISA-based capture and detectionassay against the central Fc region of the protein. The proteinconcentration of each PD-1-Fc-OX40L chimeric protein with differentjoining linker sequence (#1 to #17) was determined.

FIG. 31A to FIG. 31P show the flow cytometry profiles of PD-1-Fc-OX40Lchimeric proteins with different joining linker sequences by FACSanalysis to PD-L1 or OX40. The EC₅₀ values were calculated for eachPD-1-Fc-OX40L chimeric protein with different joining linker sequence(#2 to #17—see X-axis label and chart in the Examples below for linkeridentity).

FIG. 32 is a table showing joining linkers and Fc linkers that can becombined into illustrative modular linkers. The illustrative modularlinkers shown can be combined with any herein-described Type I and TypeII proteins and/or extracellular domains of a herein-described Type Iand Type II proteins to form a chimeric protein of the presentinvention.

DETAILED DESCRIPTION

The present invention is based, in part, on the discovery that chimericproteins can be engineered from the extracellular, or effector, regionsof immune-modulating transmembrane proteins in a manner that exploitsthe orientations of these proteins (e.g. Type I versus Type II) andtherefore allows the delivery of immune stimulatory and/or immuneinhibitory signals, including, for example, masking an immune inhibitorysignal and replacing it with an immune stimulatory signal in thetreatment of cancer, specifically, that LIGHT- and/or TIGIT-basedchimeric proteins have medical uses.

Chimeric Proteins

In some aspects, the chimeric protein is of a general structure of: Nterminus-(a)-(b)-(c)-C terminus, where (a) is a first domain comprisingan extracellular domain of a Type I transmembrane protein, (b) is alinker having at least one cysteine residue capable of forming adisulfide bond (including without limitation, hinge-CH2-CH3 Fc domain isderived from human IgG4), and (c) is a second domain comprising anextracellular domain of Type II transmembrane protein, where the linkerconnects the first domain and the second domain and optionally comprisesone or more joining linkers as described herein, where one of the firstand second extracellular domains is an immune inhibitory signal and oneof the first and second extracellular domains is an immune stimulatorysignal.

In embodiments, chimeric protein refers to a recombinant fusion protein,e.g. a single polypeptide having the extracellular domains describedherein. For example, in embodiments, the chimeric protein is translatedas a single unit in a cell. In embodiments, chimeric protein refers to arecombinant protein of multiple polypeptides, e.g. multipleextracellular domains described herein, that are linked to yield asingle unit, e.g. in vitro (e.g. with one or more synthetic linkersdescribed herein).

In embodiments, the chimeric protein is chemically synthesized as onepolypeptide or each domain may be chemically synthesized separately andthen combined. In embodiments, a portion of the chimeric protein istranslated and a portion is chemically synthesized.

In embodiments, an extracellular domain refers to a portion of atransmembrane protein which is capable of interacting with theextracellular environment. In embodiments, an extracellular domainrefers to a portion of a transmembrane protein which is sufficient tobind to a ligand or receptor and effective transmit a signal to a cell.In embodiments, an extracellular domain is the entire amino acidsequence of a transmembrane protein which is external of a cell or thecell membrane. In embodiments, an extracellular domain is the thatportion of an amino acid sequence of a transmembrane protein which isexternal of a cell or the cell membrane and is needed for signaltransduction and/or ligand binding as may be assayed using methods knowin the art (e.g. in vitro ligand binding and/or cellular activationassays).

In embodiments, an immune inhibitory signal refers to a signal thatdiminishes or eliminates an immune response. For example, in the contextof oncology, such signals may diminish or eliminate antitumor immunity.Under normal physiological conditions, inhibitory signals are useful inthe maintenance of self-tolerance (e.g. prevention of autoimmunity) andalso to protect tissues from damage when the immune system is respondingto pathogenic infection. For instance, without limitation, immuneinhibitory signal may be identified by detecting an increase in cellularproliferation, cytokine production, cell killing activity or phagocyticactivity when such an inhibitory signal is blocked.

In embodiments, an immune stimulatory signal refers to a signal thatenhances an immune response. For example, in the context of oncology,such signals may enhance antitumor immunity. For instance, withoutlimitation, immune stimulatory signal may be identified by directlystimulating proliferation, cytokine production, killing activity orphagocytic activity of leukocytes. Specific examples include directstimulation of TNF superfamily receptors such as OX40, LTbR, 4-1BB orTNFRSF25 using either receptor agonist antibodies or using chimericproteins encoding the ligands for such receptors (OX40L, LIGHT, 4-1BBL,TL1A, respectively). Stimulation from any one of these receptors maydirectly stimulate the proliferation and cytokine production ofindividual T cell subsets. Another example includes direct stimulationof an immune inhibitory cell with through a receptor that inhibits theactivity of such an immune suppressor cell. This would include, forexample, stimulation of CD4+FoxP3+ regulatory T cells with a GITRagonist antibody or GITRL containing chimeric protein, which wouldreduce the ability of those regulatory T cells to suppress theproliferation of conventional CD4+ or CD8+ T cells. In another example,this would include stimulation of CD40 on the surface of an antigenpresenting cell using a CD40 agonist antibody or a chimeric proteincontaining CD40L, causing activation of antigen presenting cellsincluding enhanced ability of those cells to present antigen in thecontext of appropriate native costimulatory molecules, including thosein the B7 or TNF superfamily. In another example, this would includestimulation of LTBR on the surface of a lymphoid or stromal cell using aLIGHT containing chimeric protein, causing activation of the lymphoidcell and/or production of pro-inflammatory cytokines or chemokines tofurther stimulate an immune response, optionally within a tumor.

Membrane proteins typically consist of an extracellular domain, one or aseries of transmembrane domains, and an intracellular domain. Withoutwishing to be bound by theory, the extracellular domain of a membraneprotein is responsible for interacting with a soluble or membrane boundreceptor or ligand. Without wishing to be bound by theory, thetrans-membrane domain(s) are responsible for localizing a protein to theplasma membrane. Without wishing to be bound by theory, theintracellular domain of a membrane protein is responsible forcoordinating interactions with cellular signaling molecules tocoordinate intracellular responses with the extracellular environment(or visa-versa). There are two types of single-pass membrane proteins,those with an extracellular amino terminus and intracellular carboxyterminus (Type I) and those with an extracellular carboxy terminus andintracellular amino terminus (Type II). Both Type I and Type II membraneproteins can be either receptors or ligands. For Type I membraneproteins, the amino terminus of the protein faces outside the cell, andtherefore contains the functional domains that are responsible forinteracting with other binding partners (either ligands or receptors) inthe extracellular environment. For Type II membrane proteins, thecarboxy terminus of the protein faces outside the cell, and thereforecontains the functional domains that are responsible for interactingwith other binding partners (either ligands or receptors) in theextracellular environment. Thus, these two types of proteins haveopposite orientations to each other.

Because the outward facing domains of Type I and Type II membraneproteins are opposite, it is possible to link the extracellular domainsof a Type I and Type II membrane protein such that the ‘outward facing’domains of the molecules are also in opposing orientation to each other(FIG. 1D). The resulting construct would therefore consist of theextracellular domain of a Type I membrane protein on the ‘left’ side ofthe molecule, connected to the extracellular domain of a Type IImembrane protein on the ‘right’ side of the molecule using a linkersequence. This construct could be produced by cloning of these threefragments (the extracellular domain of a Type I protein, followed by alinker sequence, followed by the extracellular domain of a Type IIprotein) into a vector (plasmid, viral or other) wherein the aminoterminus of the complete sequence corresponded to the ‘left’ side of themolecule containing the Type I protein and the carboxy terminus of thecomplete sequence corresponded to the ‘right’ side of the moleculecontaining the Type II protein. Accordingly, in embodiments, the presentchimeric proteins are engineered as such.

In embodiments, the extracellular domain may be used to produce asoluble protein to competitively inhibit signaling by that receptor'sligand. In embodiments, the extracellular domain may be used to provideartificial signaling.

In embodiments, the extracellular domain of a Type I transmembraneprotein is an immune inhibitory signal. In embodiments, theextracellular domain of a Type II transmembrane protein is an immunestimulatory signal.

In embodiments, the present chimeric proteins comprise an extracellulardomain of a Type I transmembrane protein, or a functional fragmentthereof. In embodiments, the present chimeric proteins comprise anextracellular domain of a Type II transmembrane protein, or a functionalfragment thereof. In embodiments, the present chimeric proteins comprisean extracellular domain of a Type I transmembrane protein, or afunctional fragment thereof, and an extracellular domain of a Type IItransmembrane protein, or a functional fragment thereof.

In embodiments, the present chimeric proteins may be engineered totarget one or more molecules that reside on human leukocytes including,without limitation, the extracellular domains (where applicable) ofSLAMF4, IL-2 R α, 4-1BB/TNFRSF9, IL-2 R β, ALCAM, BTLA, B7-1, IL-4 R,B7-H3, BLAME/SLAMFS, CEACAM1, IL-6 R, IL-7 Rα, IL-10R α, IL-I 0 R β,IL-12 R β 1, IL-12 R β 2, CD2, IL-13 R α 1, IL-13, CD3, CD4, ILT2/CDS5j,ILT3/CDS5k, ILT4/CDS5d, ILT5/CDS5a, lutegrin α 4/CD49d, CDS, Integrin αE/CD103, CD6, Integrin α M/CD 11 b, CDS, Integrin α X/CD11c, Integrin β2/CDIS, KIR/CD15S, CD27/TNFRSF7, KIR2DL1, CD2S, KIR2DL3, CD30/TNFRSFS,KIR2DL4/CD15Sd, CD31/PECAM-1, KIR2DS4, CD40 Ligand/TNFSF5, CD43, LAIR1,CD45, LAIR2, CDS3, Leukotriene B4-R1, CDS4/SLAMF5, NCAM-L1, CD94, NKG2A,CD97, NKG2C, CD229/SLAMF3, NKG2D, CD2F-10/SLAMF9, NT-4, CD69,NTB-A/SLAMF6, Common γ Chain/IL-2 R γ, Osteopontin, CRACC/SLAMF7, PD-1,CRTAM, PSGL-1, CTLA-4, RANK/TNFRSF11A, CX3CR1, CX3CL1, L-Selectin, SIRPβ 1, SLAM, TCCR/WSX-1, DNAM-1, Thymopoietin, EMMPRIN/CD147, TIM-1,EphB6, TIM-2, Fas/TNFRSF6, TIM-3, Fas Ligand/TNFSF6, TIM-4, FcγRIII/CD16, TIM-6, TNFR1/TNFRSF1A, Granulysin, TNF RIII/TNFRSF1B, TRAILRI/TNFRSFIOA, ICAM-1/CD54, TRAIL R2/TNFRSF10B, ICAM-2/CD102,TRAILR3/TNFRSF10C, IFN-γR1, TRAILR4/TNFRSF10D, IFN-γ R2, TSLP, IL-1 R1,LIGHT, LTBR (TNFRSF3) and TSLP R.

The activation of regulatory T cells is critically influenced bycostimulatory and coinhibitory signals. Two major families ofcostimulatory molecules include the B7 and the tumor necrosis factor(TNF) families. These molecules bind to receptors on T cells belongingto the CD28 or TNF receptor families, respectively. Many well-definedcoinhibitors and their receptors belong to the B7 and CD28 families.

In embodiments, the present chimeric proteins may be engineered totarget one or more molecules involved in immune inhibition, includingfor example TIGIT.

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of an immune inhibitory agent, including forexample TIGIT.

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of a Type I membrane protein which has immuneinhibitory properties. In embodiments, the chimeric protein isengineered to disrupt, block, reduce, and/or inhibit the transmission ofan immune inhibitory signal.

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of an immune stimulatory signal is LIGHT(CD258).

In embodiments, the chimeric protein simulates binding of an inhibitorysignal ligand to its cognate receptor (e.g. TIGIT to CD155/PVR,Nectin-2, Nectin-3 and/or Nectin-4) but inhibits the inhibitory signaltransmission to an immune cell (e.g. a T cell, macrophage or otherleukocyte).

In embodiments, the chimeric protein comprises an immune inhibitoryreceptor extracellular domain and an immune stimulatory ligandextracellular domain which can, without limitation, deliver an immunestimulation to a T cell while masking a tumor cell's immune inhibitorysignals. In embodiments, the chimeric protein delivers a signal that hasthe net result of T cell activation.

In embodiments, the chimeric protein comprises an immune inhibitorysignal which is an ECD of a receptor of an immune inhibitory signal andthis acts on a tumor cell that bears a cognate ligand of the immuneinhibitory signal. In embodiments, the chimeric protein comprises animmune stimulatory signal which is an ECD of a ligand of an immunestimulatory signal and this acts on a T cell that bears a cognatereceptor of the immune stimulatory signal. In embodiments, the chimericprotein comprises both (i) an immune inhibitory signal which is areceptor of an immune inhibitory signal and this acts on a tumor cellthat bears a cognate ligand of the immune inhibitory signal and (ii) animmune stimulatory signal which is a ligand of an immune stimulatorysignal and this acts on a T cell that bears a cognate receptor of theimmune stimulatory signal.

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of one or more of the immune-modulating agentsdescribed in Mahoney, Nature Reviews Drug Discovery 2015:14; 561-585,the entire contents of which are hereby incorporated by reference.

In embodiments, a chimeric protein is capable of binding murineligand(s)/receptor(s).

In embodiments, a chimeric protein is capable of binding humanligand(s)/receptor(s)

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of a Type II membrane protein which has immunestimulatory properties. In embodiments, the chimeric protein isengineered to enhance, increase, and/or stimulate the transmission of animmune stimulatory signal.

For instance, in embodiments, the extracellular domain of a Type Itransmembrane protein is from TIGIT.

TIGIT is a poliovirus receptor (PVR)-like protein, an immunoreceptorexpressed on T cells that contains immunoglobulin and immunoreceptortyrosine-based inhibitory motif (ITIM) domains. As such, TIGIT acts asan inhibitory immune checkpoint on both T cells and natural killer (NK)cells, providing an opportunity to target both the adaptive and innatearms of the immune system.

TIGIT is expressed on NK cells and subsets of activated, memory andregulatory T cells, and particularly on follicular helper T cells withinsecondary lymphoid organs CD155/PVR is up-regulated on endothelial cellsby IFN-gamma and is highly expressed on immature thymocytes, lymph nodedendritic cells, and tumor cells of epithelial and neuronal origin. Inembodiments, the present chimeric proteins (e.g. comprising the TIGITECD) modulate any of the cells described immediately above (e.g. in thecontext of an immune synapse).

TIGIT binds CD155/PVR, Nectin-2, Nectin-3 and Nectin-4. In embodiments,the present chimeric proteins (e.g. comprising the TIGIT ECD) modulatethe binding of TIGIT to CD155/PVR (e.g. reduce or disrupt the binding orsignal transmission). In embodiments, the present chimeric proteins(e.g. comprising the TIGIT ECD) modulate the binding of TIGIT toNectin-2 (e.g. reduce or disrupt the binding or signal transmission). Inembodiments, the present chimeric proteins (e.g. comprising the TIGITECD) modulate the binding of TIGIT to Nectin-3 (e.g. reduce or disruptthe binding or signal transmission). In embodiments, the presentchimeric proteins (e.g. comprising the TIGIT ECD) modulate the bindingof TIGIT to Nectin-4 (e.g. reduce or disrupt the binding or signaltransmission).

In embodiments, the chimeric protein is of a general structure of: Nterminus-(a)-(b)-(c)-C terminus, where (a) is a first domain comprisingan extracellular domain of a Type I transmembrane protein, thetransmembrane protein being TIGIT, (b) is a linker comprising at leastone cysteine residue capable of forming a disulfide bond (includingwithout limitation, hinge-CH2-CH3 Fc domain is derived from human IgG4),and (c) is a second domain comprising an extracellular domain of Type IItransmembrane protein, the transmembrane protein being selected from4-1BBL, GITRL, TL1A, and LIGHT, where the linker connects the firstdomain and the second domain and optionally comprises one or morejoining linkers as described herein.

In embodiments, the chimeric protein comprises the extracellular domainof the immune inhibitory agent TIGIT and is paired with an immunestimulatory agent as follows: TIGIT/OX-40L; TIGIT/4-1BBL, TIGIT/LIGHT;TIGIT/GITRL; TIGIT/CD70; TIGIT/CD30L; TIGIT/CD40L; TIGIT/CD137L;TIGIT/TL1A; and TIGIT/OX40L. In embodiments the chimeric protein isTIGIT-Fc-4-1BBL, TIGIT-Fc-GITRL, TIGIT-Fc-LIGHT, TIGIT-Fc-OX40L, orTIGIT-Fc-TL1A, in which the Fc represents a linker that comprises atleast a portion of an Fc domain of an antibody and which comprises atleast one cysteine residue capable of forming a disulfide bond.

For instance, in embodiments, the extracellular domain of a Type IItransmembrane protein is from LIGHT.

LIGHT (HVEM-L, TNFSF14, or CD258), an entity homologous to lymphotoxins,with inducible nature, and able to compete with herpes simplex virusglycoprotein D for herpes virus entry mediator (HVEM)/tumor necrosisfactor (TNF)-related 2 is a member of the TNF superfamily. It is a29-kDa Type II transmembrane protein, is expressed as a homotrimer onactivated T cells as well as DCs, and has three receptors, namely, HVEM,LT-β receptor (LTβR, TNFRSF3) and decoy receptor 3 (DcR3). Withoutwishing to be bound by theory, three receptors with distinct cellularexpression patterns have been known to interact with LIGHT: HVEM(TNFRSF14, CD270) detected on activated DCs, T and B cells, NK cells,monocytes, and endothelial cells; LTβR found on follicular DCs andstromal cells and binds LIGHT; and the soluble entity decoy receptor 3(DcR3) detected on diverse cancer cells such as multiple myeloma anddiffuse large B-cell lymphoma. In embodiments, the present chimericproteins can disrupt or decrease the interaction of LIGHT with one ormore of these three receptors.

LIGHT binds LTBR, and potentially HVEM as well as DcR3. In embodiments,the present chimeric proteins (e.g. comprising the LIGHT ECD) modulatethe binding of LIGHT to LTBR (e.g. increase or promote the binding orsignal transmission). LTBR is expressed by visceral, lymphoid, and otherstroma, epithelia and myeloid cells, but not lymphocytes. Inembodiments, the present chimeric proteins (e.g. comprising the LIGHTECD) modulate one or more of visceral, lymphoid, and other stroma,epithelia and myeloid cells. In embodiments, the present chimericproteins (e.g. comprising the LIGHT ECD) modulate the binding of LIGHTto HVEM (e.g. increase or promote the binding or signal transmission).In embodiments, the present chimeric proteins (e.g. comprising the LIGHTECD) modulate the binding of LIGHT to DcR3 (e.g. increase or promote thebinding or signal transmission).

In embodiments, the chimeric protein is of a general structure of: Nterminus-(a)-(b)-(c)-C terminus, where (a) is a first domain comprisingan extracellular domain of a Type I transmembrane protein, thetransmembrane protein being selected from PD-1, CD172a(SIRPα), andTIGIT, (b) is a linker comprising at least one cysteine residue capableof forming a disulfide bond (including without limitation, hinge-CH2-CH3Fc domain is derived from human IgG4), and (c) is a second domaincomprising an extracellular domain of Type II transmembrane protein, thetransmembrane protein being LIGHT, where the linker connects the firstdomain and the second domain and optionally comprises one or morejoining linkers as described herein.

In embodiments, the chimeric protein comprises the extracellular domainof the immune stimulatory agent LIGHT and is paired with an immuneinhibitory agent as follows: PD-1/LIGHT, CD172a(SIRPα)/LIGHT, andTIGIT/LIGHT. In embodiments the chimeric protein is PD-1-Fc-LIGHT,CD172a(SIRPα)-Fc-LIGHT, and TIGIT-Fc-LIGHT, in which the Fc represents alinker that comprises at least a portion of an Fc domain of an antibodyand which comprises at least one cysteine residue capable of forming adisulfide bond.

In an embodiment, the chimeric protein comprises the extracellulardomain of the immune inhibitory agent and is paired with the immunestimulatory agent. In embodiments, the chimeric protein binds to acognate receptor or ligand with a K_(D) of about 1 nM to about 5 nM, forexample, about 1 nM, about 1.5 nM, about 2 nM, about 2.5 nM, about 3 nM,about 3.5 nM, about 4 nM, about 4.5 nM, or about 5 nM. In embodiments,the chimeric protein binds to a cognate receptor or ligand with a K_(D)of about 5 nM to about 15 nM, for example, about 5 nM, about 5.5 nM,about 6 nM, about 6.5 nM, about 7 nM, about 7.5 nM, about 8 nM, about8.5 nM, about 9 nM, about 9.5 nM, about 10 nM, about 10.5 nM, about 11nM, about 11.5 nM, about 12 nM, about 12.5 nM, about 13 nM, about 13.5nM, about 14 nM, about 14.5 nM, or about 15 nM.

In embodiments, the chimeric protein exhibits enhanced stability andprotein half-life. In embodiments, the chimeric protein binds to FcRnwith high affinity. In embodiments, the chimeric protein may bind toFcRn with a K_(D) of about 1 nM to about 80 nM. For example, thechimeric protein may bind to FcRn with a K_(D) of about 1 nM, about 2nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8nM, about 9 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM,about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about55 nM, about 60 nM, about 65 nM, about 70 nM, about 71 nM, about 72 nM,about 73 nM, about 74 nM, about 75 nM, about 76 nM, about 77 nM, about78 nM, about 79 nM, or about 80 nM. In an embodiment, the chimericprotein may bind to FcRn with a K_(D) of about 9 nM. In embodiments, thechimeric protein does not substantially bind to other Fc receptors (i.e.other than FcRn) with effector function.

In embodiments, there is provided a method of treating a cancer and/orinflammatory disease (e.g. any one of those described elsewhere herein)by administering to a subject one or more of a PD-1-Fc-LIGHT,CD172a(SIRPα)-Fc-LIGHT, and TIGIT-Fc-LIGHT chimeric protein, in whichthe Fc represents a linker that comprises at least a portion of an Fcdomain of an antibody and which comprises at least one cysteine residuecapable of forming a disulfide bond. In embodiments, the methodgenerates a memory response which may, e.g. be capable of preventingrelapse. In embodiments, the method includes a sustained therapeuticeffect of the one or more of a PD-1-Fc-LIGHT, CD172a(SIRPα)-Fc-LIGHT,and TIGIT-Fc-LIGHT, e.g. due to binding of the extracellular domaincomponents to their respective binding partners with slow off rates(K_(d) or K_(off)) to optionally provide sustained negative signalmasking effect and/or a longer positive signal effect, e.g. to allow aneffector cell to be adequately stimulated for an anti-tumor effect.

In embodiments, there is provided a method of treating a cancer or aninflammatory disease (e.g. any one of those described elsewhere herein)by administering to a subject one or more of a TIGIT-Fc-4-1BBL,TIGIT-Fc-GITRL, TIGIT-Fc-TL1A, and TIGIT-Fc-LIGHT chimeric protein, inwhich the Fc represents a linker that comprises at least a portion of anFc domain of an antibody and which comprises at least one cysteineresidue capable of forming a disulfide bond. In embodiments, the methodgenerates a memory response which may, e.g. be capable of preventingrelapse. In embodiments, the method includes a sustained therapeuticeffect of the one or more of a TIGIT-Fc-4-1BBL, TIGIT-Fc-GITRL,TIGIT-Fc-TL1A, and TIGIT-Fc-LIGHT, e.g. due to binding of theextracellular domain components to their respective binding partnerswith slow off rates (K_(d) or K_(off)) to optionally provide sustainednegative signal masking effect and/or a longer positive signal effect,e.g. to allow an effector cell to be adequately stimulated for ananti-tumor effect.

In embodiments, the present chimeric proteins may comprises variants ofthe extracellular domains described herein, for instance, a sequencehaving at least about 60%, or at least about 61%, or at least about 62%,or at least about 63%, or at least about 64%, or at least about 65%, orat least about 66%, or at least about 67%, or at least about 68%, or atleast about 69%, or at least about 70%, or at least about 71%, or atleast about 72%, or at least about 73%, or at least about 74%, or atleast about 75%, or at least about 76%, or at least about 77%, or atleast about 78%, or at least about 79%, or at least about 80%, or atleast about 81%, or at least about 82%, or at least about 83%, or atleast about 84%, or at least about 85%, or at least about 86%, or atleast about 87%, or at least about 88%, or at least about 89%, or atleast about 90%, or at least about 91%, or at least about 92%, or atleast about 93%, or at least about 94%, or at least about 95%, or atleast about 96%, or at least about 97%, or at least about 98%, or atleast about 99%) sequence identity with the known amino acid or nucleicacid sequence of any of the disclosed extracellular domains, e.g. humanextracellular domains, e.g. one or more of SEQ IDs NOs: 2, 4, 7, 10, 13,16, 19, 22, 25, 27, 29, 31, 37, or 41.

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of LIGHT (SEQ ID NO: 2).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of PD-1 (SEQ ID NO: 4).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of TIGIT (SEQ ID NO: 10).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of CD172a(SIRPα) (SEQ ID NO: 7).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of LIGHT (SEQ ID NO: 2) and the extracellulardomain of PD-1 (SEQ ID NO: 4).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of LIGHT (SEQ ID NO: 2) and the extracellulardomain of TIGIT (SEQ ID NO: 10).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of LIGHT (SEQ ID NO: 2) and the extracellulardomain of CD172a(SIRPα) (SEQ ID NO: 7).

In embodiments, the chimeric protein of the present invention comprisesthe hinge-CH2-CH3 domain from a human IgG4 antibody sequence (SEQ ID NO:46, 47, or 48).

In embodiments, the chimeric protein of the present invention comprisesthe hinge-CH2-CH3 domain from a human IgG4 antibody sequence (SEQ ID NO:46, 47, or 48) and this sequence is flanked by at least one joininglinker selected from SKYGPPCPSCP (SEQ ID NO: 49), SKYGPPCPPCP (SEQ IDNO: 50), IEGRMD SEQ ID NO: 52 (optionally SKYGPPCPSCP (SEQ ID NO: 49) orSKYGPPCPPCP (SEQ ID NO: 50) is N terminal and one of IEGRMD SEQ ID NO:52 is C terminal).

In embodiments, a chimeric protein comprises a modular linker as shownin FIG. 32.

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of LIGHT and the extracellular domain of PD-1,using the hinge-CH2-CH3 domain from a human IgG4 antibody sequence as alinker (this PD-1-Fc-LIGHT chimera is SEQ ID NO: 5).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of LIGHT and the extracellular domain of TIGIT,using the hinge-CH2-CH3 domain from a human IgG4 antibody sequence as alinker (this TIGIT-Fc-LIGHT chimera is SEQ ID NO: 11).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of LIGHT and the extracellular domain ofCD172a(SIRPα), using the hinge-CH2-CH3 domain from a human IgG4 antibodysequence as a linker (this CD172a(SIRPα)-Fc-LIGHT chimera is SEQ ID NO:8).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of TIGIT (SEQ ID NO: 10).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of 4-1BBL (SEQ ID NO: 13).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of GITRL (SEQ ID NO: 16).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of TL1A (SEQ ID NO: 19).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of LIGHT (SEQ ID NO: 2).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of OX40L (SEQ ID NO: 22).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of TIGIT (SEQ ID NO: 10) and the extracellulardomain of 4-1BBL (SEQ ID NO: 13).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of TIGIT (SEQ ID NO: 10) and the extracellulardomain of GITRL (SEQ ID NO: 16).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of TIGIT (SEQ ID NO: 10) and the extracellulardomain of TL1A (SEQ ID NO: 19).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of TIGIT (SEQ ID NO: 10) and the extracellulardomain of LIGHT (SEQ ID NO: 2).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of TIGIT (SEQ ID NO: 10) and the extracellulardomain of OX40L (SEQ ID NO: 22).

In embodiments, the chimeric protein of the present invention comprisesthe hinge-CH2-CH3 domain from a human IgG4 antibody sequence (SEQ ID NO:46, 47, or 48).

In embodiments, the chimeric protein of the present invention comprisesthe hinge-CH2-CH3 domain from a human IgG4 antibody sequence (SEQ ID NO:46, 47, or 48) and this sequence is flanked by at least one joininglinker selected from SKYGPPCPSCP (SEQ ID NO: 49), SKYGPPCPPCP (SEQ IDNO: 50), IEGRMD SEQ ID NO: 52 (optionally SKYGPPCPSCP (SEQ ID NO: 49) orSKYGPPCPPCP (SEQ ID NO: 50) is N terminal and one of IEGRMD SEQ ID NO:52 is C terminal).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of TIGIT and the extracellular domain of 4-1BBL,using the hinge-CH2-CH3 domain from a human IgG4 antibody sequence as alinker (this TIGIT-Fc-4-1BBL chimera is SEQ ID NO: 14).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of TIGIT and the extracellular domain of GITRL,using the hinge-CH2-CH3 domain from a human IgG4 antibody sequence as alinker (this TIGIT-Fc-GITRL chimera is SEQ ID NO: 17).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of TIGIT and the extracellular domain of TL1A,using the hinge-CH2-CH3 domain from a human IgG4 antibody sequence as alinker (this TIGIT-Fc-TL1A chimera is SEQ ID NO: 20).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of TIGIT and the extracellular domain of LIGHT,using the hinge-CH2-CH3 domain from a human IgG4 antibody sequence as alinker (this TIGIT-Fc-LIGHT chimera is SEQ ID NO: 11).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of TIGIT and the extracellular domain of OX40L,using the hinge-CH2-CH3 domain from a human IgG4 antibody sequence as alinker (this TIGIT-Fc-OX40L chimera is SEQ ID NO: 23).

In embodiments, a chimeric protein can comprise an extracellular domainfrom a sequence identified herein combined with an extracellular domainfrom another sequence identified herein. For example, the sequence of aTIGIT-Fc-TL1A chimeric protein could include the extracellular domain ofTIGIT as disclosed above in SEQ ID NO: 10 and the extracellular domainof TL1A as disclosed above in SEQ ID NO: 19.

In embodiments, additional chimeric proteins and methods using theadditional chimeric proteins (e.g., in treating a cancer and/or treatingan inflammatory disease): TIGIT-Fc-4-1BBL, TIGIT-Fc-CD30L,TIGIT-Fc-FasL, TIGIT-Fc-GITRL, TIGIT-Fc-TL1A, and TIGIT-Fc-TRAIL. Theamino acid sequence for 4-1BBL, CD30L, FasL, GITRL, TL1A, and TRAIL,respectively, comprises SEQ ID NO: 12, 26, 30, 15, 18, and 40. The aminoacid sequence for extracellular domain of 4-1BBL, CD30L, FasL, GITRL,TL1A, and TRAIL, respectively, are SEQ ID NO: 13, 27, 31, 16, 19, and41.

In embodiments, the present chimeric proteins may be variants describedherein, for instance, the present chimeric proteins may have a sequencehaving at least about 60%, or at least about 61%, or at least about 62%,or at least about 63%, or at least about 64%, or at least about 65%, orat least about 66%, or at least about 67%, or at least about 68%, or atleast about 69%, or at least about 70%, or at least about 71%, or atleast about 72%, or at least about 73%, or at least about 74%, or atleast about 75%, or at least about 76%, or at least about 77%, or atleast about 78%, or at least about 79%, or at least about 80%, or atleast about 81%, or at least about 82%, or at least about 83%, or atleast about 84%, or at least about 85%, or at least about 86%, or atleast about 87%, or at least about 88%, or at least about 89%, or atleast about 90%, or at least about 91%, or at least about 92%, or atleast about 93%, or at least about 94%, or at least about 95%, or atleast about 96%, or at least about 97%, or at least about 98%, or atleast about 99%) sequence identity with the amino acid sequence of thepresent chimeric proteins, e.g. one or more of SEQ IDs NOs: 5, 8, 11,14, 17, 20, 23, 42, 43, 44, or 45.

In embodiments, the present chimeric proteins comprise an extracellulardomain of a human Type I transmembrane protein as recited in TABLE 1 ofPCT/US2016/054598, or a functional fragment thereof. In embodiments, thepresent chimeric proteins comprise an extracellular domain of a humanType II transmembrane protein as recited in TABLE 2 ofPCT/US2016/054598, or a functional fragment thereof. In embodiments, thepresent chimeric proteins comprise an extracellular domain of a Type Itransmembrane protein as recited in TABLE 1 of PCT/US2016/054598, or afunctional fragment thereof, and an extracellular domain of a Type IItransmembrane protein as recited in TABLE 2 of PCT/US2016/054598, or afunctional fragment thereof. The entire contents of PCT/US2016/054598are hereby incorporated by reference.

In embodiments, the chimeric protein may comprise an amino acid sequencehaving one or more amino acid mutations relative to any of the proteinsequences described herein. In embodiments, the one or more amino acidmutations may be independently selected from substitutions, insertions,deletions, and truncations.

In embodiments, the amino acid mutations are amino acid substitutions,and may include conservative and/or non-conservative substitutions.

“Conservative substitutions” may be made, for instance, on the basis ofsimilarity in polarity, charge, size, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the amino acid residuesinvolved. The 20 naturally occurring amino acids can be grouped into thefollowing six standard amino acid groups: (1) hydrophobic: Met, Ala,Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3)acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influencechain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

As used herein, “conservative substitutions” are defined as exchanges ofan amino acid by another amino acid listed within the same group of thesix standard amino acid groups shown above. For example, the exchange ofAsp by Glu retains one negative charge in the so modified polypeptide.In addition, glycine and proline may be substituted for one anotherbased on their ability to disrupt α-helices.

As used herein, “non-conservative substitutions” are defined asexchanges of an amino acid by another amino acid listed in a differentgroup of the six standard amino acid groups (1) to (6) shown above.

In embodiments, the substitutions may also include non-classical aminoacids (e.g. selenocysteine, pyrrolysine, N-formylmethionine 3-alanine,GABA and δ-Aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers ofthe common amino acids, 2,4-diaminobutyric acid, α-amino isobutyricacid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx,6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionicacid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme,citrulline, homocitrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine,fluoro-amino acids, designer amino acids such as 13 methyl amino acids,C α-methyl amino acids, N α-methyl amino acids, and amino acid analogsin general).

Mutations may also be made to the nucleotide sequences of the chimericproteins by reference to the genetic code, including taking into accountcodon degeneracy.

In embodiments, the chimeric protein comprises a linker. In embodiments,the linker comprising at least one cysteine residue capable of forming adisulfide bond. As described elsewhere herein, such at least onecysteine residue capable of forming a disulfide bond is, without wishingto be bound by theory, responsible for maintain a proper multimericstate of the chimeric protein and allowing for efficient production.

In embodiments, the linker may be derived from naturally-occurringmulti-domain proteins or are empirical linkers as described, forexample, in Chichili et al., (2013), Protein Sci. 22(2):153-167, Chen etal., (2013), Adv Drug Deliv Rev. 65(10):1357-1369, the entire contentsof which are hereby incorporated by reference. In embodiments, thelinker may be designed using linker designing databases and computerprograms such as those described in Chen et al., (2013), Adv Drug DelivRev. 65(10):1357-1369 and Crasto et. al., (2000), Protein Eng.13(5):309-312, the entire contents of which are hereby incorporated byreference.

In embodiments, the linker is a synthetic linker such as PEG.

In embodiments, the linker is a polypeptide. In embodiments, the linkeris less than about 500 amino acids long, about 450 amino acids long,about 400 amino acids long, about 350 amino acids long, about 300 aminoacids long, about 250 amino acids long, about 200 amino acids long,about 150 amino acids long, or about 100 amino acids long. For example,the linker may be less than about 100, about 95, about 90, about 85,about 80, about 75, about 70, about 65, about 60, about 55, about 50,about 45, about 40, about 35, about 30, about 25, about 20, about 19,about 18, about 17, about 16, about 15, about 14, about 13, about 12,about 11, about 10, about 9, about 8, about 7, about 6, about 5, about4, about 3, or about 2 amino acids long. In embodiments, the linker isflexible. In another embodiment, the linker is rigid.

In embodiments, the linker is substantially comprised of glycine andserine residues (e.g. about 30%, or about 40%, or about 50%, or about60%, or about 70%, or about 80%, or about 90%, or about 95%, or about97%, or about 98%, or about 99%, or about 100% glycines and serines).

In embodiments, the linker is a hinge region of an antibody (e.g., ofIgG, IgA, IgD, and IgE, inclusive of subclasses (e.g. IgG1, IgG2, IgG3,and IgG4, and IgA1 and IgA2)). The hinge region, found in IgG, IgA, IgD,and IgE class antibodies, acts as a flexible spacer, allowing the Fabportion to move freely in space. In contrast to the constant regions,the hinge domains are structurally diverse, varying in both sequence andlength among immunoglobulin classes and subclasses. For example, thelength and flexibility of the hinge region varies among the IgGsubclasses. The hinge region of IgG1 encompasses amino acids 216-231and, because it is freely flexible, the Fab fragments can rotate abouttheir axes of symmetry and move within a sphere centered at the first oftwo inter-heavy chain disulfide bridges. IgG2 has a shorter hinge thanIgG1, with 12 amino acid residues and four disulfide bridges. The hingeregion of IgG2 lacks a glycine residue, is relatively short, andcontains a rigid poly-proline double helix, stabilized by extrainter-heavy chain disulfide bridges. These properties restrict theflexibility of the IgG2 molecule. IgG3 differs from the other subclassesby its unique extended hinge region (about four times as long as theIgG1 hinge), containing 62 amino acids (including 21 prolines and 11cysteines), forming an inflexible poly-proline double helix. In IgG3,the Fab fragments are relatively far away from the Fc fragment, givingthe molecule a greater flexibility. The elongated hinge in IgG3 is alsoresponsible for its higher molecular weight compared to the othersubclasses. The hinge region of IgG4 is shorter than that of IgG1 andits flexibility is intermediate between that of IgG1 and IgG2. Theflexibility of the hinge regions reportedly decreases in the orderIgG3>IgG1>IgG4>IgG2. In embodiments, the linker may be derived fromhuman IgG4 and contain one or more mutations to enhance dimerization(including S228P) or FcRn binding.

According to crystallographic studies, the immunoglobulin hinge regioncan be further subdivided functionally into three regions: the upperhinge region, the core region, and the lower hinge region. See Shin etal., 1992 Immunological Reviews 130:87. The upper hinge region includesamino acids from the carboxyl end of CH1 to the first residue in thehinge that restricts motion, generally the first cysteine residue thatforms an interchain disulfide bond between the two heavy chains. Thelength of the upper hinge region correlates with the segmentalflexibility of the antibody. The core hinge region contains theinter-heavy chain disulfide bridges, and the lower hinge region joinsthe amino terminal end of the C_(H2) domain and includes residues inC_(H2). Id. The core hinge region of wild-type human IgG1 contains thesequence Cys-Pro-Pro-Cys which, when dimerized by disulfide bondformation, results in a cyclic octapeptide believed to act as a pivot,thus conferring flexibility. In embodiments, the present linkercomprises, one, or two, or three of the upper hinge region, the coreregion, and the lower hinge region of any antibody (e.g., of IgG, IgA,IgD, and IgE, inclusive of subclasses (e.g. IgG1, IgG2, IgG3, and IgG4,and IgA1 and IgA2)). The hinge region may also contain one or moreglycosylation sites, which include a number of structurally distincttypes of sites for carbohydrate attachment. For example, IgA1 containsfive glycosylation sites within a 17-amino-acid segment of the hingeregion, conferring resistance of the hinge region polypeptide tointestinal proteases, considered an advantageous property for asecretory immunoglobulin. In embodiments, the linker of the presentinvention comprises one or more glycosylation sites.

In embodiments, the linker comprises an Fc domain of an antibody (e.g.,of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g. IgG1, IgG2,IgG3, and IgG4, and IgA1 and IgA2)). In embodiments, the linkercomprises a hinge-CH2-CH3 Fc domain derived from a human IgG4 antibody.In embodiments, the linker comprises a hinge-CH2-CH3 Fc domain derivedfrom a human IgG1 antibody. In embodiments, the Fc domain exhibitsincreased affinity for and enhanced binding to the neonatal Fc receptor(FcRn). In embodiments, the Fc domain includes one or more mutationsthat increases the affinity and enhances binding to FcRn. Withoutwishing to be bound by theory, it is believed that increased affinityand enhanced binding to FcRn increases the in vivo half-life of thepresent chimeric proteins.

In embodiments, the Fc domain linker contains one or more amino acidsubstitutions at amino acid residue 250, 252, 254, 256, 308, 309, 311,416, 428, 433 or 434 (in accordance with Kabat numbering, as in as inKabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991) expressly incorporated herein by reference), or equivalentsthereof. In an embodiment, the amino acid substitution at amino acidresidue 250 is a substitution with glutamine. In an embodiment, theamino acid substitution at amino acid residue 252 is a substitution withtyrosine, phenylalanine, tryptophan or threonine. In an embodiment, theamino acid substitution at amino acid residue 254 is a substitution withthreonine. In an embodiment, the amino acid substitution at amino acidresidue 256 is a substitution with serine, arginine, glutamine, glutamicacid, aspartic acid, or threonine. In an embodiment, the amino acidsubstitution at amino acid residue 308 is a substitution with threonine.In an embodiment, the amino acid substitution at amino acid residue 309is a substitution with proline. In an embodiment, the amino acidsubstitution at amino acid residue 311 is a substitution with serine. Inan embodiment, the amino acid substitution at amino acid residue 385 isa substitution with arginine, aspartic acid, serine, threonine,histidine, lysine, alanine or glycine. In an embodiment, the amino acidsubstitution at amino acid residue 386 is a substitution with threonine,proline, aspartic acid, serine, lysine, arginine, isoleucine, ormethionine. In an embodiment, the amino acid substitution at amino acidresidue 387 is a substitution with arginine, proline, histidine, serine,threonine, or alanine. In an embodiment, the amino acid substitution atamino acid residue 389 is a substitution with proline, serine orasparagine. In an embodiment, the amino acid substitution at amino acidresidue 416 is a substitution with leucine. In an embodiment, the aminoacid substitution at amino acid residue 428 is a substitution withserine. In an embodiment, the amino acid substitution at amino acidresidue 433 is a substitution with arginine, serine, isoleucine,proline, or glutamine. In an embodiment, the amino acid substitution atamino acid residue 434 is a substitution with histidine, phenylalanine,or tyrosine.

In embodiments, the Fc domain linker (e.g., comprising an IgG constantregion) comprises one or more mutations such as substitutions at aminoacid residue 252, 254, 256, 433, 434, or 436 (in accordance with Kabatnumbering, as in as in Kabat, et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991) expressly incorporated hereinby reference). In an embodiment, the IgG constant region includes atriple M252Y/S254T/T256E mutation or YTE mutation. In anotherembodiment, the IgG constant region includes a triple H433K/N434F/Y436Hmutation or KFH mutation. In a further embodiment, the IgG constantregion includes an YTE and KFH mutation in combination.

In embodiments, the modified humanized antibodies of the inventioncomprise an IgG constant region that contains one or more mutations atamino acid residues 250, 253, 307, 310, 380, 416, 428, 433, 434, and435. Illustrative mutations include T250Q, M428L, T307A, E380A, 1253A,H310A, R416S, M428L, H433K, N434A, N434F, N434S, and H435A. In anembodiment, the IgG constant region comprises a M428UN434S mutation orLS mutation. In another embodiment, the IgG constant region comprises aT250Q/M428L mutation or QL mutation. In another embodiment, the IgGconstant region comprises an N434A mutation. In another embodiment, theIgG constant region comprises a T307A/E380A/N434A mutation or MAmutation. In another embodiment, the IgG constant region comprises an1253A/H310A/H435A mutation or IHH mutation. In another embodiment, theIgG constant region comprises a H433K/N434F mutation. In anotherembodiment, the IgG constant region comprises a M252Y/S254T/T256E and aH433K/N434F mutation in combination.

Additional illustrative mutations in the IgG constant region aredescribed, for example, in Robbie, et al., Antimicrobial Agents andChemotherapy (2013), 57(12):6147-6153, Dall'Acqua et al., JBC (2006),281(33):23514-24, Dall'Acqua et al., Journal of Immunology (2002),169:5171-80, Ko et al. Nature (2014) 514:642-645, Grevys et al. Journalof Immunology. (2015), 194(11):5497-508, and U.S. Pat. No. 7,083,784,the entire contents of which are hereby incorporated by reference.

In embodiments, the linker comprises the amino acid sequence of SEQ IDNO: 46, or at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identitythereto. In embodiments, mutations are made to SEQ ID NO: 46 to increasestability and/or half-life. For instance, in embodiments, the linker hasthe amino acid sequence of SEQ ID NO: 47, or at least 90%, or 93%, or95%, or 97%, or 98%, or 99% identity thereto. In embodiments, the linkercomprises the amino acid sequence of SEQ ID NO: 48, or at least 90%, or93%, or 95%, or 97%, or 98%, or 99% identity thereto.

Without wishing to be bound by theory, including a linker comprising atleast a part of an Fc domain in a chimeric protein, helps avoidformation of insoluble and, likely, non-functional protein concatamersand/or aggregates. This is in part due to the presence of cysteines inthe Fc domain which are capable of forming disulfide bonds betweenchimeric proteins.

An illustrative Fc stabilizing mutant is S228P. Illustrative Fchalf-life extending mutants are T250Q, M428L, V308T, L309P, and Q311Sand the present linkers may comprise 1, or 2, or 3, or 4, or 5 of thesemutants.

Further, one or more joining linkers may be employed to connect an Fcdomain in a linker (e.g., one of SEQ ID NO: 46, SEQ ID NO: 47, or SEQ IDNO: 48 or at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identitythereto) and the extracellular domains. For example, any one of SEQ IDNO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQID NO: 54, or variants thereof may connect an extracellular domain asdescribed herein and a linker as described herein. Optionally, any oneof SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ IDNO: 53, SEQ ID NO: 54, or variants thereof are displaced between anextracellular domain as described herein and a linker as describedherein. Optionally, any one of SEQ ID NOs: 49 to 95, or variants thereofare located between an extracellular domain as described herein and anFc domain as described herein. In embodiments, a chimeric proteincomprises one joining linker preceding an Fc domain and a second joininglinker following the Fc domain; thus, a chimeric protein may comprisethe following structure:

-   -   ECD 1-Joining Linker 1-Fc Domain-Joining Linker 2-ECD 2.

In embodiments, the first and second joining linkers may be different orthey may be the same.

The amino acid sequences of illustrative linkers are provided in Table 1below:

TABLE 1 Illustrative linkers (Fc domain linkers and joining linkers)SEQ ID NO. Sequence 46APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSSWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 47APEFLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTTPHSDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSSWQEGNVFSCSVLHEALHNHYTQKSLSLSLGK 48APEFLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGK 49 SKYGPPCPSCP 50 SKYGPPCPPCP 51SKYGPP 52 IEGRMD 53 GGGVPRDCG 54 IEGRMDGGGGAGGGG 55 GGGSGGGS 56GGGSGGGGSGGG 57 EGKSSGSGSESKST 58 GGSG 59 GGSGGGSGGGSG 60EAAAKEAAAKEAAAK 61 EAAAREAAAREAAAREAAAR 62 GGGGSGGGGSGGGGSAS 63GGGGAGGGG 64 GS or GGS or LE 65 GSGSGS 66 GSGSGSGSGS 67 GGGGSAS 68APAPAPAPAPAPAPAPAPAP 69 CPPC 70 GGGGS 71 GGGGSGGGGS 72 GGGGSGGGGSGGGGS73 GGGGSGGGGSGGGGSGGGGS 74 GGGGSGGGGSGGGGSGGGGSGGGGS 75GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 76 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 77GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 78 GGSGGSGGGGSGGGGS 79 GGGGGGGG80 GGGGGG 81 EAAAK 82 EAAAKEAAAK 83 EAAAKEAAAKEAAAK 84 AEAAAKEAAAKA 85AEAAAKEAAAKEAAAKA 86 AEAAAKEAAAKEAAAKEAAAKA 87AEAAAKEAAAKEAAAKEAAAKEAAAKA 88AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA 89 PAPAP 90KESGSVSSEQLAQFRSLD 91 GSAGSAAGSGEF 92 GGGSE 93 GSESG 94 GSEGS 95GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS

Additional illustrative joining linkers include, but are not limited to,linkers having the sequence LE, GGGGS (SEQ ID NO: 70), (GGGGS)_(n)(n=1-4) (SEQ ID NO: 70-73), (Gly)₈ (SEQ ID NO: 79), (Gly)₆ (SEQ ID NO:80), (EAAAK)_(n) (n=1-3) (SEQ ID NO: 81-83), A(EAAAK)_(n)A (n=2-5) (SEQID NO: 84-87), AEAAAKEAAAKA (SEQ ID NO: 84), A(EAAAK)₄ALEA(EAAAK)₄A (SEQID NO: 88), PAPAP (SEQ ID NO: 89), KESGSVSSEQLAQFRSLD (SEQ ID NO: 90),EGKSSGSGSESKST (SEQ ID NO: 57), GSAGSAAGSGEF (SEQ ID NO: 91), and(XP)_(n), with X designating any amino acid, e.g., Ala, Lys, or Glu.

In embodiments, the joining linker is substantially comprised of glycineand serine residues (e.g., about 30%, or about 40%, or about 50%, orabout 60%, or about 70%, or about 80%, or about 90%, or about 95%, orabout 97%, or about 98%, or about 99%, or about 100% glycines andserines). For example, in embodiments, the joining linker is(Gly₄Ser)_(n), where n is from about 1 to about 8, e.g., 1, 2, 3, 4, 5,6, 7, or 8 (SEQ ID NO: 70 to SEQ ID NO: 77, respectively). Inembodiments, the joining linker sequence is GGSGGSGGGGSGGGGS (SEQ ID NO:78). Additional illustrative joining linkers include, but are notlimited to, linkers having the sequence LE, (Gly)₈ (SEQ ID NO: 79),(Gly)₆ (SEQ ID NO: 80), (EAAAK)_(n) (n=1-3) (SEQ ID NO: 81-SEQ ID NO:83), A(EAAAK)_(n)A (n=2-5) (SEQ ID NO: 84-SEQ ID NO: 87),A(EAAAK)₄ALEA(EAAAK)₄A (SEQ ID NO: 88), PAPAP (SEQ ID NO: 89),KESGSVSSEQLAQFRSLD (SEQ ID NO: 90), GSAGSAAGSGEF (SEQ ID NO: 91), and(XP)_(n), with X designating any amino acid, e.g., Ala, Lys, or Glu. Inembodiments, the joining linker is GGS.

In embodiments, the joining linker is one or more of GGGSE (SEQ ID NO:92), GSESG (SEQ ID NO: 93), GSEGS (SEQ ID NO: 94),GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS (SEQ ID NO: 95), and a joininglinker of randomly placed G, S, and E every 4 amino acid intervals.

In embodiments, a chimeric protein comprises a modular linker as shownin FIG. 32.

In embodiments, the linker may be flexible, including without limitationhighly flexible. In embodiments, the linker may be rigid, includingwithout limitation a rigid alpha helix.

In embodiments, the linker may be functional. For example, withoutlimitation, the linker may function to improve the folding and/orstability, improve the expression, improve the pharmacokinetics, and/orimprove the bioactivity of the present chimeric protein. In anotherexample, the linker may function to target the chimeric protein to aparticular cell type or location.

In embodiments, the present chimeric proteins are capable of, and can beused in methods comprising, promoting immune activation (e.g. againsttumors). In embodiments, the present chimeric proteins are capable of,and can be used in methods comprising, suppressing immune inhibition(e.g. that allows tumors to survive). In embodiments, the presentchimeric proteins provide improved immune activation and/or improvedsuppression of immune inhibition due to the proximity of signaling thatis provided by the chimeric nature of the constructs.

In embodiments, the present chimeric proteins are capable of, or can beused in methods comprising, modulating the amplitude of an immuneresponse, e.g. modulating the level of effector output. In embodiments,e.g. when used for the treatment of cancer, the present chimericproteins alter the extent of immune stimulation as compared to immuneinhibition to increase the amplitude of a T cell response, including,without limitation, stimulating increased levels of cytokine production,proliferation or target killing potential.

In embodiments the present chimeric proteins, in embodiments are capableof, or find use in methods involving, masking an inhibitory ligand onthe surface of a tumor cell and replacing that immune inhibitory ligandwith an immune stimulatory ligand. Accordingly, the present chimericproteins, in embodiments are capable of, or find use in methodsinvolving, reducing or eliminating an inhibitory immune signal and/orincreasing or activating an immune stimulatory signal. For example, atumor cell bearing an inhibitory signal (and thus evading an immuneresponse) may be substituted for a positive signal binding on a T cellthat can then attack a tumor cell. Accordingly, in embodiments, aninhibitory immune signal is masked by the present constructs and astimulatory immune signal is activated. Such beneficial properties areenhanced by the single construct approach of the present chimericproteins. For instance, the signal replacement can be effected nearlysimultaneously and the signal replacement is tailored to be local at asite of clinical importance (e.g. the tumor microenvironment). Furtherembodiments apply the same principle to other chimeric proteinconstructs, such as, for example, (i) the extracellular domain of TIGITand (ii) extracellular domain of 4-1BBL; (i) the extracellular domain ofTIGIT and (ii) extracellular domain of GITRL; (i) the extracellulardomain of TIGIT and (ii) extracellular domain of TL1A; (i) theextracellular domain of TIGIT and (ii) extracellular domain of LIGHT;and (i) the extracellular domain of PD-1 and (ii) extracellular domainof LIGHT; and (i) the extracellular domain of CD172a(SIRPα) and (ii)extracellular domain of LIGHT; and (i) the extracellular domain of TIGITand (ii) extracellular domain of LIGHT; among others.

In embodiments, the present chimeric proteins are capable of, or finduse in methods comprising, stimulating or enhancing the binding ofimmune stimulatory receptor/ligand pairs. Illustrative T cellcostimulatory receptors and their ligands include OX-40:OX40-L,CD27:CD70, CD30:CD30-L, CD40:CD40-L; CD137:CD137-L, HVEM:LIGHT,GITR:GITR-L, TNFRSF25:TL1A, DR5:TRAIL, and BTLA:HVEM. In embodiments,the present chimeric proteins are capable of, or find use in methodscomprising, inhibiting or reducing the binding of immune inhibitoryreceptor/ligand pairs. Illustrative T cell coinhibitory receptors andtheir ligands include, for example, CTLA-4:CD80/CD86, PD-1:PD-L1/PD-L2,BTLA:HVEM, TIM-3:galectin-9/phosphatidylserine, TIGIT/CD155 or CD112,VISTANSIG8, CD172a(SIRPα)/CD47, B7H3R/B7H3, B7H4R/B7H4, CD244/CD48,TMIGD2/HHLA2, among others.

In embodiments, the present chimeric protein blocks, reduces and/orinhibits PD-1 and PD-L1 or PD-L2 and/or the binding of PD-1 with PD-L1or PD-L2. In embodiments, the present chimeric protein blocks, reducesand/or inhibits the activity of CTLA-4 and/or the binding of CTLA-4 withone or more of AP2M1, CD80, CD86, SHP-2, and PPP2R5A. In embodiments,the present chimeric protein increases and/or stimulates GITR and/or thebinding of GITR with one or more of GITR ligand. In embodiments, thepresent chimeric protein increases and/or stimulates OX40 and/or thebinding of OX40 with one or more of OX40 ligand.

In embodiments, the present chimeric proteins are capable of, or finduse in methods involving, enhancing, restoring, promoting and/orstimulating immune modulation. In embodiments, the present chimericproteins described herein, restore, promote and/or stimulate theactivity or activation of one or more immune cells against tumor cellsincluding, but not limited to: T cells, cytotoxic T lymphocytes, Thelper cells, natural killer (NK) cells, natural killer T (NKT) cells,anti-tumor macrophages (e.g. M1 macrophages), B cells, and dendriticcells. In embodiments, the present chimeric proteins enhance, restore,promote and/or stimulate the activity and/or activation of T cells,including, by way of a non-limiting example, activating and/orstimulating one or more T-cell intrinsic signals, including apro-survival signal; an autocrine or paracrine growth signal; a p38MAPK-, ERK-, STAT-, JAK-, AKT- or PI3K-mediated signal; ananti-apoptotic signal; and/or a signal promoting and/or necessary forone or more of: proinflammatory cytokine production or T cell migrationor T cell tumor infiltration.

In embodiments, the present chimeric proteins are capable of, or finduse in methods involving, causing an increase of one or more of T cells(including without limitation cytotoxic T lymphocytes, T helper cells,natural killer T (NKT) cells), B cells, natural killer (NK) cells,natural killer T (NKT) cells, dendritic cells, monocytes, andmacrophages (e.g. one or more of M1 and M2) into a tumor or the tumormicroenvironment. In embodiments, the present chimeric proteins arecapable of, or find use in methods involving, inhibiting and/or causinga decrease in recruitment of immunosuppressive cells (e.g.myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs),tumor associated neutrophils (TANs), M2 macrophages, and tumorassociated macrophages (TAMs)) to the tumor and/or tumormicroenvironment (TME). In embodiments, the present therapies may alterthe ratio of M1 versus M2 macrophages in the tumor site and/or TME tofavor M1 macrophages.

In embodiments, the present chimeric proteins are capable of, and can beused in methods comprising, inhibiting and/or reducing T cellinactivation and/or immune tolerance to a tumor, comprisingadministering an effective amount of a chimeric protein described hereinto a subject. In embodiments, the present chimeric proteins are able toincrease the serum levels of various cytokines including, but notlimited to, one or more of IFNγ, TNFα, IL-2, IL-4, IL-5, IL-6, IL-9,IL-10, IL-13, IL-17A, IL-17F, and IL-22. In embodiments, the presentchimeric proteins are capable of enhancing IL-2, IL-4, IL-5, IL-10,IL-13, IL-17A, IL-22, TNFα or IFNγ in the serum of a treated subject. Inembodiments, administration of the present chimeric protein is capableof enhancing TNFα secretion. In a specific embodiment, administration ofthe present chimeric protein is capable of enhancing superantigenmediated TNFα secretion by leukocytes. Detection of such a cytokineresponse may provide a method to determine the optimal dosing regimenfor the indicated chimeric protein.

In embodiments, the present chimeric proteins inhibit, block and/orreduce cell death of an anti-tumor CD8+ and/or CD4+ T cell; orstimulate, induce, and/or increase cell death of a pro-tumor T cell. Tcell exhaustion is a state of T cell dysfunction characterized byprogressive loss of proliferative and effector functions, culminating inclonal deletion. Accordingly, a pro-tumor T cell refers to a state of Tcell dysfunction that arises during many chronic infections and cancer.This dysfunction is defined by poor proliferative and/or effectorfunctions, sustained expression of inhibitory receptors and atranscriptional state distinct from that of functional effector ormemory T cells. Exhaustion prevents optimal control of infection andtumors. In addition, an anti-tumor CD8+ and/or CD4+ T cell refers to Tcells that can mount an immune response to a tumor. Illustrativepro-tumor T cells include, but are not limited to, Tregs, CD4+ and/orCD8+ T cells expressing one or more checkpoint inhibitory receptors, Th2cells and Th17 cells. Checkpoint inhibitory receptors refers toreceptors expressed on immune cells that prevent or inhibit uncontrolledimmune responses.

In embodiments, the present chimeric proteins are capable of, and can beused in methods comprising, increasing a ratio of effector T cells toregulatory T cells. Illustrative effector T cells include ICOS⁺ effectorT cells; cytotoxic T cells (e.g. αβ TCR, CD3⁺, CD8⁺, CD45RO⁺); CD4⁺effector T cells (e.g. αβ TCR, CD3⁺, CD4⁺, CCR7⁺, CD62Lhi,IL⁻7R/CD127⁺); CD8⁺ effector T cells (e.g. αβ TCR, CD3⁺, CD8⁺, CCR7⁺,CD62Lhi, IL7R/CD127⁺); effector memory T cells (e.g. CD62Llow, CD44⁺,TCR, CD3⁺, IL⁻7R/CD127⁺, IL-15R⁺, CCR7low); central memory T cells (e.g.CCR7⁺, CD62L⁺, CD27⁺; or CCR7hi, CD44⁺, CD62Lhi, TCR, CD3⁺,IL-7R/CD127⁺, IL-15R⁺); CD62L⁺ effector T cells; CD8⁺ effector memory Tcells (TEM) including early effector memory T cells (CD27⁺ CD62L⁻) andlate effector memory T cells (CD27⁻ CD62L⁻) (TemE and TemL,respectively); CD127(⁺)CD25(low/−) effector T cells; CD127(⁻) CD25(⁻)effector T cells; CD8⁺ stem cell memory effector cells (TSCM) (e.g.CD44(low)CD62L(high)CD122(high)sca(⁺)); TH1 effector T-cells (e.g.CXCR3⁺, CXCR6⁺ and CCR5⁺; or αβ TCR, CD3⁺, CD4⁺, IL-12R⁺, IFNγR⁺,CXCR3⁺), TH2 effector T cells (e.g. CCR3⁺, CCR4⁺ and CCR8⁺; or αβ TCR,CD3⁺, CD4⁺, IL-4R⁺, IL-33R⁺, CCR4⁺, IL-17RB⁺, CRTH2⁺); TH9 effector Tcells (e.g. αβ TCR, CD3⁺, CD4⁻); TH17 effector T cells (e.g. αβ TCR,CD3⁺, CD4⁺, IL-23R⁺, CCR6⁺, IL-1R⁺); CD4⁺CD45RO⁺CCR7⁺ effector T cells,CD4⁺CD45RO⁺CCR7(⁻) effector T cells; and effector T cells secretingIL-2, IL-4 and/or IFN-γ. Illustrative regulatory T cells include ICOS⁺regulatory T cells, CD4⁺CD25⁺FOXP3⁺ regulatory T cells, CD4⁺CD25⁺regulatory T cells, CD4⁺CD25⁻ regulatory T cells, CD4⁺CD25highregulatory T cells, TIM-3⁺PD-1⁺ regulatory T cells, lymphocyteactivation gene-3 (LAG-3)⁺ regulatory T cells, CTLA-4/CD152⁺ regulatoryT cells, neuropilin-1 (Nrp-1)⁺ regulatory T cells, CCR4⁺CCR8⁺ regulatoryT cells, CD62L (L-selectin)′ regulatory T cells, CD45RBlow regulatory Tcells, CD127low regulatory T cells, LRRC32/GARP⁺ regulatory T cells,CD39⁺ regulatory T cells, GITR⁺ regulatory T cells, LAP⁺ regulatory Tcells, 1B11⁺ regulatory T cells, BTLA⁺ regulatory T cells, type 1regulatory T cells (Tr1 cells),T helper type 3 (Th3) cells, regulatorycell of natural killer T cell phenotype (NKTregs), CD8⁺ regulatory Tcells, CD8⁺CD28⁻ regulatory T cells and/or regulatory T-cells secretingIL-10, IL-35, TGF-β, TNF-α, Galectin-1, IFN-γ and/or MCP1.

In embodiments, the chimeric protein generates a memory response whichmay, e.g., be capable of preventing relapse or protecting the animalfrom a rechallenge. Thus, an animal treated with the chimeric protein islater able to attack tumor cells and/or prevent development of tumorswhen rechallenged after an initial treatment with the chimeric protein.Accordingly, a chimeric protein of the present invention stimulates bothactive tumor destruction and also immune recognition of tumor antigens,which are essential in programming a memory response capable ofpreventing relapse.

In embodiments, the present chimeric proteins are capable of, and can beused in methods comprising, transiently stimulating effector T cells forno longer than about 12 hours, about 24 hours, about 48 hours, about 72hours or about 96 hours or about 1 week or about 2 weeks. Inembodiments, the present chimeric proteins are capable of, and can beused in methods comprising, transiently depleting or inhibitingregulatory T cells for no longer than about 12 hours, about 24 hours,about 48 hours, about 72 hours or about 96 hours or about 1 week orabout 2 weeks. In embodiments, the transient stimulation of effector Tcells and/or transient depletion or inhibition of regulatory T cellsoccurs substantially in a patient's bloodstream or in a particulartissue/location including lymphoid tissues such as for example, the bonemarrow, lymph-node, spleen, thymus, mucosa-associated lymphoid tissue(MALT), non-lymphoid tissues, or in the tumor microenvironment.

In embodiments, the present chimeric proteins provide advantagesincluding, without limitation, ease of use and ease of production. Thisis because two distinct immunotherapy agents are combined into a singleproduct which allows for a single manufacturing process instead of twoindependent manufacturing processes. In addition, administration of asingle agent instead of two separate agents allows for easieradministration and greater patient compliance. Further, in contrast to,for example, monoclonal antibodies, which are large multimeric proteinscontaining numerous disulfide bonds and post-translational modificationssuch as glycosylation, the present chimeric proteins are easier and morecost effective to manufacture.

In embodiments, the present chimeric protein is producible in amammalian host cell as a secretable and fully functional singlepolypeptide chain.

In embodiments, the present chimeric protein unexpectedly providesbinding of the extracellular domain components to their respectivebinding partners with slow off rates (K_(d) or K_(off)). In embodiments,this provides an unexpectedly long interaction of the receptor to ligandand vice versa. Such an effect allows for a sustained negative signalmasking effect. Further, in embodiments, this delivers a longer positivesignal effect, e.g. to allow an effector cell to be adequatelystimulated for an anti-tumor effect. For example, the present chimericprotein, e.g. via the long off rate binding allows sufficient signaltransmission to provide T cell proliferation and allow for anti-tumorattack. By way of further example, the present chimeric protein, e.g.via the long off rate binding allows sufficient signal transmission toprovide release of stimulatory signals, such as, for example, cytokines.

The stable synapse of cells promoted by the present agents (e.g. a tumorcell bearing negative signals and a T cell which could attack the tumor)provides spatial orientation to favor tumor reduction—such aspositioning the T cells to attack tumor cells and/or stericallypreventing the tumor cell from delivering negative signals, includingnegative signals beyond those masked by the chimeric protein of theinvention.

In embodiments, this provides longer on-target (e.g. intra-tumoral)half-life (t_(1/2)) as compared to serum t_(1/2) of the chimericproteins. Such properties could have the combined advantage of reducingoff-target toxicities which may be associated with systemic distributionof the chimeric proteins.

In embodiments, the present agents allow certain immune cells to act,e.g. in an antitumoral manner, by preventing and/or disruptinginhibition of NK cells and/or subsets of activated, memory and/orregulatory T cells, and/or helper T cells by blocking a signal via TIGITand, optionally, create further immune responses via 4-1BBL-, and/orGITRL-, and/or TL1A-, and/or LIGHT-based stimulatory signaling.

In embodiments, the present agents allow certain immune cells to act,e.g. in an antitumoral manner, by stimulating and/or increasingstimulatory LIGHT-based signaling, e.g. on visceral and/or lymphoidand/or other stroma and/or epithelia and/or myeloid cells and,optionally, create further immune responses via blockade or reduction ofPD-1-, and/or CD172a(SIRPα)-, and/or TIGIT-based inhibitory signaling.

Further, in embodiments, the present chimeric proteins providesynergistic therapeutic effects as it allows for improved site-specificinterplay of two immunotherapy agents.

In embodiments, the present chimeric proteins provide the potential forreducing off-site and/or systemic toxicity.

In embodiments, the present chimeric proteins provide reducedside-effects, e.g., GI complications, relative to currentimmunotherapies, e.g., antibodies directed to checkpoint molecules asdescribed herein. Illustrative GI complications include abdominal pain,appetite loss, autoimmune effects, constipation, cramping, dehydration,diarrhea, eating problems, fatigue, flatulence, fluid in the abdomen orascites, gastrointestinal (GI) dysbiosis, GI mucositis, inflammatorybowel disease, irritable bowel syndrome (IBS-D and IBS-C), nausea, pain,stool or urine changes, ulcerative colitis, vomiting, weight gain fromretaining fluid, and/or weakness.

Diseases; Methods of Treatment, and Patient Selections

In embodiments, the present invention pertains to cancers and/or tumors;for example, the treatment or prevention of cancers and/or tumors. Asdescribed elsewhere herein, the treatment of cancer may involve inembodiments, modulating the immune system with the present chimericproteins to favor immune stimulation over immune inhibition.

Cancers or tumors refer to an uncontrolled growth of cells and/orabnormal increased cell survival and/or inhibition of apoptosis whichinterferes with the normal functioning of the bodily organs and systems.Included are benign and malignant cancers, polyps, hyperplasia, as wellas dormant tumors or micrometastases. Also, included are cells havingabnormal proliferation that is not impeded by the immune system (e.g.virus infected cells). The cancer may be a primary cancer or ametastatic cancer. The primary cancer may be an area of cancer cells atan originating site that becomes clinically detectable, and may be aprimary tumor. In contrast, the metastatic cancer may be the spread of adisease from one organ or part to another non-adjacent organ or part.The metastatic cancer may be caused by a cancer cell that acquires theability to penetrate and infiltrate surrounding normal tissues in alocal area, forming a new tumor, which may be a local metastasis. Thecancer may also be caused by a cancer cell that acquires the ability topenetrate the walls of lymphatic and/or blood vessels, after which thecancer cell is able to circulate through the bloodstream (thereby beinga circulating tumor cell) to other sites and tissues in the body. Thecancer may be due to a process such as lymphatic or hematogeneousspread. The cancer may also be caused by a tumor cell that comes to restat another site, re-penetrates through the vessel or walls, continues tomultiply, and eventually forms another clinically detectable tumor. Thecancer may be this new tumor, which may be a metastatic (or secondary)tumor.

The cancer may be caused by tumor cells that have metastasized, whichmay be a secondary or metastatic tumor. The cells of the tumor may belike those in the original tumor. As an example, if a breast cancer orcolon cancer metastasizes to the liver, the secondary tumor, whilepresent in the liver, is made up of abnormal breast or colon cells, notof abnormal liver cells. The tumor in the liver may thus be a metastaticbreast cancer or a metastatic colon cancer, not liver cancer.

The cancer may have an origin from any tissue. The cancer may originatefrom melanoma, colon, breast, or prostate, and thus may be made up ofcells that were originally skin, colon, breast, or prostate,respectively. The cancer may also be a hematological malignancy, whichmay be leukemia or lymphoma. The cancer may invade a tissue such asliver, lung, bladder, or intestinal.

Representative cancers and/or tumors of the present invention include,but are not limited to, a basal cell carcinoma, biliary tract cancer;bladder cancer; bone cancer; brain and central nervous system cancer;breast cancer; cancer of the peritoneum; cervical cancer;choriocarcinoma; colon and rectum cancer; connective tissue cancer;cancer of the digestive system; endometrial cancer; esophageal cancer;eye cancer; cancer of the head and neck; gastric cancer (includinggastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma;intra-epithelial neoplasm; kidney or renal cancer; larynx cancer;leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer,non-small cell lung cancer, adenocarcinoma of the lung, and squamouscarcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavitycancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreaticcancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectalcancer; cancer of the respiratory system; salivary gland carcinoma;sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicularcancer; thyroid cancer; uterine or endometrial cancer; cancer of theurinary system; vulval cancer; lymphoma including Hodgkin's andnon-Hodgkin's lymphoma, as well as B-cell lymphoma (including lowgrade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL)NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL;high grade immunoblastic NHL; high grade lymphoblastic NHL; high gradesmall non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; as well as othercarcinomas and sarcomas; and post-transplant lymphoproliferativedisorder (PTLD), as well as abnormal vascular proliferation associatedwith phakomatoses, edema (such as that associated with brain tumors),and Meigs' syndrome.

In embodiments, the chimeric protein is used to treat a subject that hasa treatment-refractory cancer. In embodiments, the chimeric protein isused to treat a subject that is refractory to one or moreimmune-modulating agents. For example, in embodiments, the chimericprotein is used to treat a subject that presents no response totreatment, or even progress, after 12 weeks or so of treatment. Forinstance, in embodiments, the subject is refractory to a PD-1 and/orPD-L1 and/or PD-L2 agent, including, for example, nivolumab(ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB),pembrolizumab (KEYTRUDA, MERCK), pidilizumab (CT-011, CURE TECH),MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), Ibrutinib(PHARMACYCLICS/ABBVIE), atezolizumab (TECENTRIQ, GENENTECH), and/orMPDL3280A (ROCHE)-refractory patients. For instance, in embodiments, thesubject is refractory to an anti-CTLA-4 agent, e.g. ipilimumab(YERVOY)-refractory patients (e.g. melanoma patients). Accordingly, inembodiments the present invention provides methods of cancer treatmentthat rescue patients that are non-responsive to various therapies,including monotherapy of one or more immune-modulating agents.

In embodiments, the present invention provides chimeric proteins whichtarget a cell or tissue within the tumor microenviroment. Inembodiments, the cell or tissue within the tumor microenvironmentexpresses one or more targets or binding partners of the chimericprotein. The tumor microenvironment refers to the cellular milieu,including cells, secreted proteins, physiological small molecules, andblood vessels in which the tumor exists. In embodiments, the cells ortissue within the tumor microenvironment are one or more of: tumorvasculature; tumor-infiltrating lymphocytes; fibroblast reticular cells;endothelial progenitor cells (EPC); cancer-associated fibroblasts;pericytes; other stromal cells; components of the extracellular matrix(ECM); dendritic cells; antigen presenting cells; T-cells; regulatory Tcells; macrophages; neutrophils; and other immune cells located proximalto a tumor. In embodiments, the present chimeric protein targets acancer cell. In embodiments, the cancer cell expresses one or more oftargets or binding partners of the chimeric protein.

In embodiments, the chimeric protein of the invention may target a cell(e.g., cancer cell or immune cell) that expresses TIGIT. In embodiments,the chimeric protein of the invention may target a cell (e.g., cancercell or immune cell) that expresses CD155/PVR. In embodiments, thechimeric protein of the invention may target a cell (e.g., cancer cellor immune cell) that expresses Nectin-2. In embodiments, the chimericprotein of the invention may target a cell (e.g., cancer cell or immunecell) that expresses Nectin-3. In embodiments, the chimeric protein ofthe invention may target a cell (e.g., cancer cell or immune cell) thatexpresses Nectin-4.

In embodiments, the chimeric protein of the invention may target a cell(e.g., cancer cell or immune cell) that expresses LIGHT. In embodiments,the chimeric protein of the invention may target a cell (e.g., cancercell or immune cell) that expresses LTBR. In embodiments, the chimericprotein of the invention may target a cell (e.g., cancer cell or immunecell) that expresses HVEM. In embodiments, the chimeric protein of theinvention may target a cell (e.g., cancer cell or immune cell) thatexpresses DcR3.

In embodiments, the present methods provide treatment with the chimericprotein in a patient who is refractory to an additional agent, such“additional agents” being described elsewhere herein, inclusive, withoutlimitation, of the various chemotherapeutic agents described herein.

In embodiments, the chimeric proteins are used to treat, control orprevent one or more inflammatory diseases or conditions. Non-limitingexamples of inflammatory diseases include acne vulgaris, acuteinflammation, allergic rhinitis, asthma, atherosclerosis, atopicdermatitis, autoimmune disease, autoinflammatory diseases, autosomalrecessive spastic ataxia, bronchiectasis, celiac disease, chroniccholecystitis, chronic inflammation, chronic prostatitis, colitis,diverticulitis, familial eosinophilia (fe), glomerulonephritis, glycerolkinase deficiency, hidradenitis suppurativa, hypersensitivities,inflammation, inflammatory bowel diseases, inflammatory pelvic disease,interstitial cystitis, laryngeal inflammatory disease, Leigh syndrome,lichen planus, mast cell activation syndrome, mastocytosis, ocularinflammatory disease, otitis, pain, pelvic inflammatory disease,reperfusion injury, respiratory disease, restenosis, rheumatic fever,rheumatoid arthritis, rhinitis, sarcoidosis, septic shock, silicosis andother pneumoconioses, transplant rejection, tuberculosis, andvasculitis.

In embodiments, the inflammatory disease is an autoimmune disease orcondition, such as multiple sclerosis, diabetes mellitus, lupus, celiacdisease, Crohn's disease, ulcerative colitis, Guillain-Barre syndrome,scleroderms, Goodpasture's syndrome, Wegener's granulomatosis,autoimmune epilepsy, Rasmussen's encephalitis, Primary biliarysclerosis, Sclerosing cholangitis, Autoimmune hepatitis, Addison'sdisease, Hashimoto's thyroiditis, Fibromyalgia, Menier's syndrome;transplantation rejection (e.g., prevention of allograft rejection)pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus,dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiplesclerosis, myasthenia gravis, Reiter's syndrome, Grave's disease, andother autoimmune diseases.

In some aspects, the present chimeric agents are used to eliminateintracellular pathogens. In some aspects, the present chimeric agentsare used to treat one or more infections. In embodiments, the presentchimeric proteins are used in methods of treating viral infections(including, for example, HIV and HCV), parasitic infections (including,for example, malaria), and bacterial infections. In embodiments, theinfections induce immunosuppression. For example, HIV infections oftenresult in immunosuppression in the infected subjects. Accordingly, asdescribed elsewhere herein, the treatment of such infections mayinvolve, in embodiments, modulating the immune system with the presentchimeric proteins to favor immune stimulation over immune inhibition.Alternatively, the present invention provides methods for treatinginfections that induce immunoactivation. For example, intestinalhelminth infections have been associated with chronic immune activation.In these embodiments, the treatment of such infections may involvemodulating the immune system with the present chimeric proteins to favorimmune inhibition over immune stimulation.

In embodiments, the present invention provides methods of treating viralinfections including, without limitation, acute or chronic viralinfections, for example, of the respiratory tract, of papilloma virusinfections, of herpes simplex virus (HSV) infection, of humanimmunodeficiency virus (HIV) infection, and of viral infection ofinternal organs such as infection with hepatitis viruses. Inembodiments, the viral infection is caused by a virus of familyFlaviviridae. In embodiments, the virus of family Flaviviridae isselected from Yellow Fever Virus, West Nile virus, Dengue virus,Japanese Encephalitis Virus, St. Louis Encephalitis Virus, and HepatitisC Virus. In embodiments, the viral infection is caused by a virus offamily Picornaviridae, e.g., poliovirus, rhinovirus, coxsackievirus. Inembodiments, the viral infection is caused by a member ofOrthomyxoviridae, e.g., an influenza virus. In embodiments, the viralinfection is caused by a member of Retroviridae, e.g., a lentivirus. Inembodiments, the viral infection is caused by a member ofParamyxoviridae, e.g., respiratory syncytial virus, a humanparainfluenza virus, rubulavirus (e.g., mumps virus), measles virus, andhuman metapneumovirus. In embodiments, the viral infection is caused bya member of Bunyaviridae, e.g., hantavirus. In embodiments, the viralinfection is caused by a member of Reoviridae, e.g., a rotavirus.

In embodiments, the present invention provides methods of treatingparasitic infections such as protozoan or helminths infections. Inembodiments, the parasitic infection is by a protozoan parasite. Inembodiments, the oritiziab parasite is selected from intestinalprotozoa, tissue protozoa, or blood protozoa. Illustrative protozoanparasites include, but are not limited to, Entamoeba hystolytica,Giardia lamblia, Cryptosporidium muris, Trypanosomatida gambiense,Trypanosomatida rhodesiense, Trypanosomatida crusi, Leishmania mexicana,Leishmania braziliensis, Leishmania tropica, Leishmania donovani,Toxoplasma gondii, Plasmodium vivax, Plasmodium ovale, Plasmodiummalariae, Plasmodium falciparum, Trichomonas vaginalis, and Histomonasmeleagridis. In embodiments, the parasitic infection is by a helminthicparasite such as nematodes (e.g., Adenophorea). In embodiments, theparasite is selected from Secementea (e.g., Trichuris trichiura, Ascarislumbricoides, Enterobius vermicularis, Ancylostoma duodenale, Necatoramericanus, Strongyloides stercoralis, Wuchereria bancrofti, Dracunculusmedinensis). In embodiments, the parasite is selected from trematodes(e.g. blood flukes, liver flukes, intestinal flukes, and lung flukes).In embodiments, the parasite is selected from: Schistosoma mansoni,Schistosoma haematobium, Schistosoma japonicum, Fasciola hepatica,Fasciola gigantica, Heterophyes, Paragonimus westermani. In in Table 1,the parasite is selected from cestodes (e.g., Taenia solium, Taeniasaginata, Hymenolepis nana, Echinococcus granulosus).

In embodiments, the present invention provides methods of treatingbacterial infections. In embodiments, the bacterial infection is bygram-positive bacteria, gram-negative bacteria, aerobic and/or anaerobicbacteria. In embodiments, the bacteria is selected from, but not limitedto, Staphylococcus, Lactobacillus, Streptococcus, Sarcina, Escherichia,Enterobacter, Klebsiella, Pseudomonas, Acinetobacter, Mycobacterium,Proteus, Campylobacter, Citrobacter, Nisseria, Baccillus, Bacteroides,Peptococcus, Clostridium, Salmonella, Shigella, Serratia, Haemophilus,Brucella and other organisms. In embodiments, the bacteria is selectedfrom, but not limited to, Pseudomonas aeruginosa, Pseudomonasfluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes,Pseudomonas putida, Stenotrophomonas maltophilia, Burkholderia cepacia,Aeromonas hydrophilia, Escherichia coli, Citrobacter freundii,Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi,Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri,Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes,Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens,Francisella tularensis, Morganella morganii, Proteus mirabilis, Proteusvulgaris, Providencia alcalifaciens, Providencia rettgeri, Providenciastuartii, Acinetobacter baumannii, Acinetobacter calcoaceticus,Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis,Yersinia pseudotuberculosis, Yersinia intermedia, Bordetella pertussis,Bordetella parapertussis, Bordetella bronchiseptica, Haemophilusinfluenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus,Haemophilus parahaemolyticus, Haemophilus ducreyi, Pasteurellamultocida, Pasteurella haemolytica, Branhamella catarrhalis,Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni,Campylobacter coli, Borrelia burgdorferi, Vibrio cholerae, Vibrioparahaemolyticus, Legionella pneumophila, Listeria monocytogenes,Neisseria gonorrhoeae, Neisseria meningitidis, Kingella, Moraxella,Gardnerella vaginalis, Bacteroides fragilis, Bacteroides distasonis,Bacteroides 3452A homology group, Bacteroides vulgatus, Bacteroidesovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroideseggerthii, Bacteroides splanchnicus, Clostridium difficile,Mycobacterium tuberculosis, Mycobacterium avium, Mycobacteriumintracellulare, Mycobacterium leprae, Corynebacterium diphtheriae,Corynebacterium ulcerans, Streptococcus pneumoniae, Streptococcusagalactiae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcusfaecium, Staphylococcus aureus, Staphylococcus epidermidis,Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcushyicus subsp. hyicus, Staphylococcus haemolyticus, Staphylococcushominis, or Staphylococcus saccharolyticus.

In some aspects, the present chimeric agents are used to treat one ormore autoimmune diseases or disorders. In embodiments, the treatment ofan autoimmune disease or disorder may involve modulating the immunesystem with the present chimeric proteins to favor immune inhibitionover immune stimulation. Illustrative autoimmune diseases or disorderstreatable with the present chimeric proteins include those in which thebody's own antigens become targets for an immune response, such as, forexample, rheumatoid arthritis, systemic lupus erythematosus, diabetesmellitus, ankylosing spondylitis, Sjōgren's syndrome, inflammatory boweldiseases (e.g. colitis ulcerosa, Crohn's disease), multiple sclerosis,sarcoidosis, psoriasis, Grave's disease, Hashimoto's thyroiditis,psoriasis, hypersensitivity reactions (e.g., allergies, hay fever,asthma, and acute edema cause Type I hypersensitivity reactions), andvasculitis.

In still another other aspect, the present invention is directed towardmethods of treating and preventing T cell-mediated diseases anddisorders, such as, but not limited to diseases or disorders describedelsewhere herein and inflammatory disease or disorder, graft-versus-hostdisease (GVHD), transplant rejection, and T cell proliferative disorder.

In some aspects, the present chimeric agents are used in methods ofactivating a T cell, e.g. via the extracellular domain having an immunestimulatory signal.

In some aspects, the present chimeric agents are used in methods ofpreventing the cellular transmission of an immunosuppressive signal.

Combination Therapies and Conjugation

In embodiments, the invention provides for chimeric proteins and methodsthat further comprise administering an additional agent to a subject. Inembodiments, the invention pertains to co-administration and/orco-formulation. Any of the compositions described herein may beco-formulated and/or co-administered.

In embodiments, any chimeric protein described herein actssynergistically when co-administered with another agent and isadministered at doses that are lower than the doses commonly employedwhen such agents are used as monotherapy. In embodiments, any agentreferenced herein may be used in combination with any of the chimericproteins described herein.

In embodiments, inclusive of, without limitation, cancer applications,the present invention pertains to chemotherapeutic agents as additionalagents. Examples of chemotherapeutic agents include, but are not limitedto, alkylating agents such as thiotepa and CYTOXAN cyclosphosphamide;alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(e.g., bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; cally statin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (e.g., cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammall and calicheamicinomegall (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCINdoxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as minoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; demecolcine; diaziquone;elformithine; elliptinium acetate; an epothilone; etoglucid; galliumnitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such asmaytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOLpaclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANECremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), andTAXOTERE doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; NAVELBINE. vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar,CPT-11) (including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); lapatinib (TYKERB); inhibitors of PKC-α, Raf, H-Ras,EGFR (e.g., erlotinib (Tarceva)) and VEGF-A that reduce cellproliferation and pharmaceutically acceptable salts, acids orderivatives of any of the above. In addition, the methods of treatmentcan further include the use of radiation. In addition, the methods oftreatment can further include the use of photodynamic therapy.

In embodiments, inclusive of, without limitation, cancer applications,the present additional agent is one or more immune-modulating agentsselected from an agent that blocks, reduces and/or inhibits PD-1 andPD-L1 or PD-L2 and/or the binding of PD-1 with PD-L1 or PD-L2 (by way ofnon-limiting example, one or more of nivolumab (ONO-4538/BMS-936558,MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, Merck),MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), atezolizumab(TECENTRIQ, GENENTECH), MPDL3280A (ROCHE)), an agent that increasesand/or stimulates CD137 (4-1BB) and/or the binding of CD137 (4-1BB) withone or more of 4-1BB ligand (by way of non-limiting example, urelumab(BMS-663513 and anti-4-1BB antibody), and an agent that blocks, reducesand/or inhibits the activity of CTLA-4 and/or the binding of CTLA-4 withone or more of AP2M1, CD80, CD86, SHP-2, and PPP2R5A and/or the bindingof OX40 with OX40L (by way of non-limiting example GBR 830 (GLENMARK),MED16469 (MEDIMMUNE).

In embodiments, inclusive of, without limitation, infectious diseaseapplications, the present invention pertains to anti-infectives asadditional agents. In embodiments, the anti-infective is an anti-viralagent including, but not limited to, Abacavir, Acyclovir, Adefovir,Amprenavir, Atazanavir, Cidofovir, Darunavir, Delavirdine, Didanosine,Docosanol, Efavirenz, Elvitegravir, Emtricitabine, Enfuvirtide,Etravirine, Famciclovir, and Foscarnet. In embodiments, theanti-infective is an anti-bacterial agent including, but not limited to,cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil,cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, andceftobiprole); fluoroquinolone antibiotics (cipro, Levaquin, floxin,tequin, avelox, and norflox); tetracycline antibiotics (tetracycline,minocycline, oxytetracycline, and doxycycline); penicillin antibiotics(amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin,vancomycin, and methicillin); monobactam antibiotics (aztreonam); andcarbapenem antibiotics (ertapenem, doripenem, imipenem/cilastatin, andmeropenem). In embodiments, the anti-infectives include anti-malarialagents (e.g., chloroquine, quinine, mefloquine, primaquine, doxycycline,artemether/lumefantrine, atovaquone/proguanil andsulfadoxine/pyrimethamine), metronidazole, tinidazole, ivermectin,pyrantel pamoate, and albendazole.

In embodiments, inclusive, without limitation, of autoimmuneapplications, the additional agent is an immunosuppressive agent. Inembodiments, the immunosuppressive agent is an anti-inflammatory agentsuch as a steroidal anti-inflammatory agent or a non-steroidalanti-inflammatory agent (NSAID). Steroids, particularly the adrenalcorticosteroids and their synthetic analogues, are well known in theart. Examples of corticosteroids useful in the present inventioninclude, without limitation, hydroxyltriamcinolone, alpha-methyldexamethasone, beta-methyl betamethasone, beclomethasone dipropionate,betamethasone benzoate, betamethasone dipropionate, betamethasonevalerate, clobetasol valerate, desonide, desoxymethasone, dexamethasone,diflorasone diacetate, diflucortolone valerate, fluadrenolone,fluclorolone acetonide, flumethasone pivalate, fluosinolone acetonide,fluocinonide, flucortine butylester, fluocortolone, fluprednidene(fluprednylidene) acetate, flurandrenolone, halcinonide, hydrocortisoneacetate, hydrocortisone butyrate, methylprednisolone, triamcinoloneacetonide, cortisone, cortodoxone, flucetonide, fludrocortisone,difluorosone diacetate, fluradrenolone acetonide, medrysone, amcinafel,amcinafide, betamethasone and the balance of its esters,chloroprednisone, clocortelone, clescinolone, dichlorisone,difluprednate, flucloronide, flunisolide, fluoromethalone, fluperolone,fluprednisolone, hydrocortisone, meprednisone, paramethasone,prednisolone, prednisone, beclomethasone dipropionate. (NSAIDS) that maybe used in the present invention, include but are not limited to,salicylic acid, acetyl salicylic acid, methyl salicylate, glycolsalicylate, salicylmides, benzyl-2,5-diacetoxybenzoic acid, ibuprofen,fulindac, naproxen, ketoprofen, etofenamate, phenylbutazone, andindomethacin. In embodiments, the immunosuppressive agent may becytostatics such as alkylating agents, antimetabolites (e.g.,azathioprine, methotrexate), cytotoxic antibiotics, antibodies (e.g.,basiliximab, daclizumab, and muromonab), anti-immunophilins (e.g.,cyclosporine, tacrolimus, sirolimus), inteferons, opioids, TNF bindingproteins, mycophenolates, and small biological agents (e.g., fingolimod,myriocin).

In embodiments, the chimeric proteins (and/or additional agents)described herein, include derivatives that are modified, i.e., by thecovalent attachment of any type of molecule to the composition such thatcovalent attachment does not prevent the activity of the composition.For example, but not by way of limitation, derivatives includecomposition that have been modified by, inter alia, glycosylation,lipidation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications can be carried out by known techniques,including, but not limited to specific chemical cleavage, acetylation,formylation, metabolic synthesis of turicamycin, etc. Additionally, thederivative can contain one or more non-classical amino acids. In stillother embodiments, the chimeric proteins (and/or additional agents)described herein further comprise a cytotoxic agent, comprising, inillustrative embodiments, a toxin, a chemotherapeutic agent, aradioisotope, and an agent that causes apoptosis or cell death. Suchagents may be conjugated to a composition described herein.

The chimeric proteins (and/or additional agents) described herein maythus be modified post-translationally to add effector moieties such aschemical linkers, detectable moieties such as for example fluorescentdyes, enzymes, substrates, bioluminescent materials, radioactivematerials, and chemiluminescent moieties, or functional moieties such asfor example streptavidin, avidin, biotin, a cytotoxin, a cytotoxicagent, and radioactive materials.

Formulations

The chimeric proteins (and/or additional agents) described herein canpossess a sufficiently basic functional group, which can react with aninorganic or organic acid, or a carboxyl group, which can react with aninorganic or organic base, to form a pharmaceutically acceptable salt. Apharmaceutically acceptable acid addition salt is formed from apharmaceutically acceptable acid, as is well known in the art. Suchsalts include the pharmaceutically acceptable salts listed in, forexample, Journal of Pharmaceutical Science, 66, 2-19 (1977) and TheHandbook of Pharmaceutical Salts; Properties, Selection, and Use. P. H.Stahl and C. G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, whichare hereby incorporated by reference in their entirety.

In embodiments, the compositions described herein are in the form of apharmaceutically acceptable salt.

Further, any chimeric protein (and/or additional agents) describedherein can be administered to a subject as a component of a compositionthat comprises a pharmaceutically acceptable carrier or vehicle. Suchcompositions can optionally comprise a suitable amount of apharmaceutically acceptable excipient so as to provide the form forproper administration. Pharmaceutical excipients can be liquids, such aswater and oils, including those of petroleum, animal, vegetable, orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. The pharmaceutical excipients can be, for example,saline, gum acacia, gelatin, starch paste, talc, keratin, colloidalsilica, urea and the like. In addition, auxiliary, stabilizing,thickening, lubricating, and coloring agents can be used. In oneembodiment, the pharmaceutically acceptable excipients are sterile whenadministered to a subject. Water is a useful excipient when any agentdescribed herein is administered intravenously. Saline solutions andaqueous dextrose and glycerol solutions can also be employed as liquidexcipients, specifically for injectable solutions. Suitablepharmaceutical excipients also include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. Any agent describedherein, if desired, can also comprise minor amounts of wetting oremulsifying agents, or pH buffering agents.

In embodiments, the compositions described herein are resuspended in asaline buffer (including, without limitation TBS, PBS, and the like).

In embodiments, the chimeric proteins may by conjugated and/or fusedwith another agent to extend half-life or otherwise improvepharmacodynamic and pharmacokinetic properties. In embodiments, thechimeric proteins may be fused or conjugated with one or more of PEG,XTEN (e.g., as rPEG), polysialic acid (POLYXEN), albumin (e.g., humanserum albumin or HAS), elastin-like protein (ELP), PAS, HAP, GLK, CTP,transferrin, and the like. In embodiments, each of the individualchimeric proteins is fused to one or more of the agents described inBioDrugs (2015) 29:215-239, the entire contents of which are herebyincorporated by reference.

Administration, Dosing, and Treatment Regimens

The present invention includes the described chimeric protein (and/oradditional agents) in various formulations. Any chimeric protein (and/oradditional agents) described herein can take the form of solutions,suspensions, emulsion, drops, tablets, pills, pellets, capsules,capsules containing liquids, powders, sustained-release formulations,suppositories, emulsions, aerosols, sprays, suspensions, or any otherform suitable for use. DNA or RNA constructs encoding the proteinsequences may also be used. In one embodiment, the composition is in theform of a capsule (see, e.g., U.S. Pat. No. 5,698,155). Other examplesof suitable pharmaceutical excipients are described in Remington'sPharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed.1995), incorporated herein by reference.

Where necessary, the formulations comprising the chimeric protein(and/or additional agents) can also include a solubilizing agent. Also,the agents can be delivered with a suitable vehicle or delivery deviceas known in the art. Combination therapies outlined herein can beco-delivered in a single delivery vehicle or delivery device.Compositions for administration can optionally include a localanesthetic such as, for example, lignocaine to lessen pain at the siteof the injection.

The formulations comprising the chimeric protein (and/or additionalagents) of the present invention may conveniently be presented in unitdosage forms and may be prepared by any of the methods well known in theart of pharmacy. Such methods generally include the step of bringingtherapeutic agents into association with a carrier, which constitutesone or more accessory ingredients. Typically, the formulations areprepared by uniformly and intimately bringing therapeutic agent intoassociation with a liquid carrier, a finely divided solid carrier, orboth, and then, if necessary, shaping the product into dosage forms ofthe desired formulation (e.g., wet or dry granulation, powder blends,etc., followed by tableting using conventional methods known in the art)

In one embodiment, any chimeric protein (and/or additional agents)described herein is formulated in accordance with routine procedures asa composition adapted for a mode of administration described herein.

Routes of administration include, for example: intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, oral, sublingual, intranasal, intracerebral, intravaginal,transdermal, rectally, by inhalation, or topically, particularly to theears, nose, eyes, or skin. In embodiments, the administering is effectedorally or by parenteral injection. In most instances, administrationresults in the release of any agent described herein into thebloodstream.

Any chimeric protein (and/or additional agents) described herein can beadministered orally. Such chimeric proteins (and/or additional agents)can also be administered by any other convenient route, for example, byintravenous infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and can be administered together with anotherbiologically active agent. Administration can be systemic or local.Various delivery systems are known, e.g., encapsulation in liposomes,microparticles, microcapsules, capsules, etc., and can be used toadminister.

In specific embodiments, it may be desirable to administer locally tothe area in need of treatment. In one embodiment, for instance in thetreatment of cancer, the chimeric protein (and/or additional agents) areadministered in the tumor microenvironment (e.g. cells, molecules,extracellular matrix and/or blood vessels that surround and/or feed atumor cell, inclusive of, for example, tumor vasculature;tumor-infiltrating lymphocytes; fibroblast reticular cells; endothelialprogenitor cells (EPC); cancer-associated fibroblasts; pericytes; otherstromal cells; components of the extracellular matrix (ECM); dendriticcells; antigen presenting cells; T-cells; regulatory T cells;macrophages; neutrophils; and other immune cells located proximal to atumor) or lymph node and/or targeted to the tumor microenvironment orlymph node. In embodiments, for instance in the treatment of cancer, thechimeric protein (and/or additional agents) are administeredintratumorally.

In the various embodiments, the present chimeric protein allows for adual effect that provides less side effects than are seen inconventional immunotherapy (e.g. treatments with one or more of OPDIVO,KEYTRUDA, YERVOY, and TECENTRIQ). For example, the present chimericproteins reduce or prevent commonly observed immune-related adverseevents that affect various tissues and organs including the skin, thegastrointestinal tract, the kidneys, peripheral and central nervoussystem, liver, lymph nodes, eyes, pancreas, and the endocrine system;such as hypophysitis, colitis, hepatitis, pneumonitis, rash, andrheumatic disease. Further, the present local administration, e.g.intratumorally, obviate adverse event seen with standard systemicadministration, e.g. IV infusions, as are used with conventionalimmunotherapy (e.g. treatments with one or more of OPDIVO, KEYTRUDA,YERVOY, and TECENTRIQ).

Dosage forms suitable for parenteral administration (e.g. intravenous,intramuscular, intraperitoneal, subcutaneous and intra-articularinjection and infusion) include, for example, solutions, suspensions,dispersions, emulsions, and the like. They may also be manufactured inthe form of sterile solid compositions (e.g. lyophilized composition),which can be dissolved or suspended in sterile injectable mediumimmediately before use. They may contain, for example, suspending ordispersing agents known in the art.

The dosage of any chimeric protein (and/or additional agents) describedherein as well as the dosing schedule can depend on various parameters,including, but not limited to, the disease being treated, the subject'sgeneral health, and the administering physician's discretion. Anychimeric protein described herein, can be administered prior to (e.g., 5minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before),concurrently with, or subsequent to (e.g., 5 minutes, 15 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of anadditional agent, to a subject in need thereof. In embodiments anychimeric protein and additional agent described herein are administered1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hourapart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart,3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hoursapart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11hours to 12 hours apart, 1 day apart, 2 days apart, 3 days apart, 4 daysapart, 5 days apart, 6 days apart, 1 week apart, 2 weeks apart, 3 weeksapart, or 4 weeks apart.

In embodiments, the present invention relates to the co-administrationof a chimeric protein which induces an innate immune response andanother chimeric protein which induces an adaptive immune response. Insuch embodiments, the chimeric protein which induces an innate immuneresponse may be administered before, concurrently with, or subsequent toadministration of the chimeric protein which induces an adaptive immuneresponse. For example, the chimeric proteins may be administered 1minute apart, 10 minutes apart, 30 minutes apart, less than 1 hourapart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart,3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hoursapart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11hours to 12 hours apart, 1 day apart, 2 days apart, 3 days apart, 4 daysapart, 5 days apart, 6 days apart, 1 week apart, 2 weeks apart, 3 weeksapart, or 4 weeks apart. In an illustrative embodiment, the chimericprotein which induces an innate immune response and the chimeric proteinwhich induces an adaptive response are administered 1 week apart, oradministered on alternate weeks (i.e., administration of the chimericprotein inducing an innate immune response is followed 1 week later withadministration of the chimeric protein which induces an adaptive immuneresponse and so forth).

The dosage of any chimeric protein (and/or additional agents) describedherein can depend on several factors including the severity of thecondition, whether the condition is to be treated or prevented, and theage, weight, and health of the subject to be treated. Additionally,pharmacogenomic (the effect of genotype on the pharmacokinetic,pharmacodynamic or efficacy profile of a therapeutic) information abouta particular subject may affect dosage used. Furthermore, the exactindividual dosages can be adjusted somewhat depending on a variety offactors, including the specific combination of the agents beingadministered, the time of administration, the route of administration,the nature of the formulation, the rate of excretion, the particulardisease being treated, the severity of the disorder, and the anatomicallocation of the disorder. Some variations in the dosage can be expected.

For administration of any chimeric protein (and/or additional agents)described herein by parenteral injection, the dosage may be about 0.1 mgto about 250 mg per day, about 1 mg to about 20 mg per day, or about 3mg to about 5 mg per day. Generally, when orally or parenterallyadministered, the dosage of any agent described herein may be about 0.1mg to about 1500 mg per day, or about 0.5 mg to about 10 mg per day, orabout 0.5 mg to about 5 mg per day, or about 200 to about 1,200 mg perday (e.g., about 200 mg, about 300 mg, about 400 mg, about 500 mg, about600 mg, about 700 mg, about 800 mg, about 900 mg, about 1,000 mg, about1,100 mg, about 1,200 mg per day).

In embodiments, administration of the chimeric protein (and/oradditional agents) described herein is by parenteral injection at adosage of about 0.1 mg to about 1500 mg per treatment, or about 0.5 mgto about 10 mg per treatment, or about 0.5 mg to about 5 mg pertreatment, or about 200 to about 1,200 mg per treatment (e.g., about 200mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700mg, about 800 mg, about 900 mg, about 1,000 mg, about 1,100 mg, about1,200 mg per treatment).

In embodiments, a suitable dosage of the chimeric protein (and/oradditional agents) is in a range of about 0.01 mg/kg to about 100 mg/kgof body weight, or about 0.01 mg/kg to about 10 mg/kg of body weight ofthe subject, for example, about 0.01 mg/kg, about 0.02 mg/kg, about 0.03mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg,about 1.1 mg/kg, about 1.2 mg/kg, about 1.3 mg/kg, about 1.4 mg/kg,about 1.5 mg/kg, about 1.6 mg/kg, about 1.7 mg/kg, about 1.8 mg/kg, 1.9mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kgbody weight, inclusive of all values and ranges therebetween.

In another embodiment, delivery can be in a vesicle, in particular aliposome (see Langer, 1990, Science 249:1527-1533; Treat et al., inLiposomes in therapy of Infectious Disease and Cancer, Lopez-Beresteinand Fidler (eds.), Liss, New York, pp. 353-365 (1989).

Any chimeric protein (and/or additional agents) described herein can beadministered by controlled-release or sustained-release means or bydelivery devices that are well known to those of ordinary skill in theart. Examples include, but are not limited to, those described in U.S.Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719;5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476;5,354,556; and 5,733,556, each of which is incorporated herein byreference in its entirety. Such dosage forms can be useful for providingcontrolled- or sustained-release of one or more active ingredientsusing, for example, hydropropylmethyl cellulose, other polymer matrices,gels, permeable membranes, osmotic systems, multilayer coatings,microparticles, liposomes, microspheres, or a combination thereof toprovide the desired release profile in varying proportions. Controlled-or sustained-release of an active ingredient can be stimulated byvarious conditions, including but not limited to, changes in pH, changesin temperature, stimulation by an appropriate wavelength of light,concentration or availability of enzymes, concentration or availabilityof water, or other physiological conditions or compounds.

In another embodiment, polymeric materials can be used (see MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug ProductDesign and Performance, Smolen and Ball (eds.), Wiley, New York (1984);Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61;see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann.Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105).

In another embodiment, a controlled-release system can be placed inproximity of the target area to be treated, thus requiring only afraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).Other controlled-release systems discussed in the review by Langer,1990, Science 249:1527-1533) may be used.

Administration of any chimeric protein (and/or additional agents)described herein can, independently, be one to four times daily or oneto four times per month or one to six times per year or once every two,three, four or five years. Administration can be for the duration of oneday or one month, two months, three months, six months, one year, twoyears, three years, and may even be for the life of the subject.

The dosage regimen utilizing any chimeric protein (and/or additionalagents) described herein can be selected in accordance with a variety offactors including type, species, age, weight, sex and medical conditionof the subject; the severity of the condition to be treated; the routeof administration; the renal or hepatic function of the subject; thepharmacogenomic makeup of the individual; and the specific compound ofthe invention employed. Any chimeric protein (and/or additional agents)described herein can be administered in a single daily dose, or thetotal daily dosage can be administered in divided doses of two, three orfour times daily. Furthermore, any chimeric protein (and/or additionalagents) described herein can be administered continuously rather thanintermittently throughout the dosage regimen.

Cells and Nucleic Acids

In embodiments, the present invention provides an expression vector,comprising a nucleic acid encoding the chimeric protein describedherein. In embodiments, the expression vector comprises DNA or RNA. Inembodiments, the expression vector is a mammalian expression vector.

Both prokaryotic and eukaryotic vectors can be used for expression ofthe chimeric protein. Prokaryotic vectors include constructs based on E.coli sequences (see, e.g., Makrides, Microbiol Rev 1996, 60:512-538).Non-limiting examples of regulatory regions that can be used forexpression in E. coli include lac, trp, Ipp, phoA, recA, tac, T3, T7 andλP_(L). Non-limiting examples of prokaryotic expression vectors mayinclude the λgt vector series such as λgt11 (Huynh et al., in “DNACloning Techniques, Vol. I: A Practical Approach,” 1984, (D. Glover,ed.), pp. 49-78, IRL Press, Oxford), and the pET vector series (Studieret al., Methods Enzymol 1990, 185:60-89). Prokaryotic host-vectorsystems cannot perform much of the post-translational processing ofmammalian cells, however. Thus, eukaryotic host-vector systems may beparticularly useful. A variety of regulatory regions can be used forexpression of the chimeric proteins in mammalian host cells. Forexample, the SV40 early and late promoters, the cytomegalovirus (CMV)immediate early promoter, and the Rous sarcoma virus long terminalrepeat (RSV-LTR) promoter can be used. Inducible promoters that may beuseful in mammalian cells include, without limitation, promotersassociated with the metallothionein II gene, mouse mammary tumor virusglucocorticoid responsive long terminal repeats (MMTV-LTR), the3-interferon gene, and the hsp70 gene (see, Williams et al., Cancer Res1989, 49:2735-42; and Taylor et al., Mol Cell Biol 1990, 10:165-75).Heat shock promoters or stress promoters also may be advantageous fordriving expression of the chimeric proteins in recombinant host cells.

In embodiments, expression vectors of the invention comprise a nucleicacid encoding the chimeric proteins (and/or additional agents), or acomplement thereof, operably linked to an expression control region, orcomplement thereof, that is functional in a mammalian cell. Theexpression control region is capable of driving expression of theoperably linked blocking and/or stimulating agent encoding nucleic acidsuch that the blocking and/or stimulating agent is produced in a humancell transformed with the expression vector.

Expression control regions are regulatory polynucleotides (sometimesreferred to herein as elements), such as promoters and enhancers, thatinfluence expression of an operably linked nucleic acid. An expressioncontrol region of an expression vector of the invention is capable ofexpressing operably linked encoding nucleic acid in a human cell. In anembodiment, the cell is a tumor cell. In another embodiment, the cell isa non-tumor cell. In an embodiment, the expression control regionconfers regulatable expression to an operably linked nucleic acid. Asignal (sometimes referred to as a stimulus) can increase or decreaseexpression of a nucleic acid operably linked to such an expressioncontrol region. Such expression control regions that increase expressionin response to a signal are often referred to as inducible. Suchexpression control regions that decrease expression in response to asignal are often referred to as repressible. Typically, the amount ofincrease or decrease conferred by such elements is proportional to theamount of signal present; the greater the amount of signal, the greaterthe increase or decrease in expression.

In an embodiment, the present invention contemplates the use ofinducible promoters capable of effecting high level of expressiontransiently in response to a cue. For example, when in the proximity ofa tumor cell, a cell transformed with an expression vector for thechimeric protein (and/or additional agents) comprising such anexpression control sequence is induced to transiently produce a highlevel of the agent by exposing the transformed cell to an appropriatecue. Illustrative inducible expression control regions include thosecomprising an inducible promoter that is stimulated with a cue such as asmall molecule chemical compound. Particular examples can be found, forexample, in U.S. Pat. Nos. 5,989,910, 5,935,934, 6,015,709, and6,004,941, each of which is incorporated herein by reference in itsentirety.

Expression control regions and locus control regions include full-lengthpromoter sequences, such as native promoter and enhancer elements, aswell as subsequences or polynucleotide variants which retain all or partof full-length or non-variant function. As used herein, the term“functional” and grammatical variants thereof, when used in reference toa nucleic acid sequence, subsequence or fragment, means that thesequence has one or more functions of native nucleic acid sequence(e.g., non-variant or unmodified sequence).

As used herein, “operable linkage” refers to a physical juxtaposition ofthe components so described as to permit them to function in theirintended manner. In the example of an expression control element inoperable linkage with a nucleic acid, the relationship is such that thecontrol element modulates expression of the nucleic acid. Typically, anexpression control region that modulates transcription is juxtaposednear the 5′ end of the transcribed nucleic acid (i.e., “upstream”).Expression control regions can also be located at the 3′ end of thetranscribed sequence (i.e., “downstream”) or within the transcript(e.g., in an intron). Expression control elements can be located at adistance away from the transcribed sequence (e.g., 100 to 500, 500 to1000, 2000 to 5000, or more nucleotides from the nucleic acid). Aspecific example of an expression control element is a promoter, whichis usually located 5′ of the transcribed sequence. Another example of anexpression control element is an enhancer, which can be located 5′ or 3′of the transcribed sequence, or within the transcribed sequence.

Expression systems functional in human cells are well known in the art,and include viral systems. Generally, a promoter functional in a humancell is any DNA sequence capable of binding mammalian RNA polymerase andinitiating the downstream (3′) transcription of a coding sequence intomRNA. A promoter will have a transcription initiating region, which isusually placed proximal to the 5′ end of the coding sequence, andtypically a TATA box located 25-30 base pairs upstream of thetranscription initiation site. The TATA box is thought to direct RNApolymerase II to begin RNA synthesis at the correct site. A promoterwill also typically contain an upstream promoter element (enhancerelement), typically located within 100 to 200 base pairs upstream of theTATA box. An upstream promoter element determines the rate at whichtranscription is initiated and can act in either orientation. Ofparticular use as promoters are the promoters from mammalian viralgenes, since the viral genes are often highly expressed and have a broadhost range. Examples include the SV40 early promoter, mouse mammarytumor virus LTR promoter, adenovirus major late promoter, herpes simplexvirus promoter, and the CMV promoter.

Typically, transcription termination and polyadenylation sequencesrecognized by mammalian cells are regulatory regions located 3′ to thetranslation stop codon and thus, together with the promoter elements,flank the coding sequence. The 3′ terminus of the mature mRNA is formedby site-specific post-translational cleavage and polyadenylation.Examples of transcription terminator and polyadenylation signals includethose derived from SV40. Introns may also be included in expressionconstructs.

There are a variety of techniques available for introducing nucleicacids into viable cells. Techniques suitable for the transfer of nucleicacid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, polymer-based systems,DEAE-dextran, viral transduction, the calcium phosphate precipitationmethod, etc. For in vivo gene transfer, a number of techniques andreagents may also be used, including liposomes; natural polymer-baseddelivery vehicles, such as chitosan and gelatin; viral vectors are alsosuitable for in vivo transduction. In some situations, it is desirableto provide a targeting agent, such as an antibody or ligand specific fora tumor cell surface membrane protein. Where liposomes are employed,proteins which bind to a cell surface membrane protein associated withendocytosis may be used for targeting and/or to facilitate uptake, e.g.,capsid proteins or fragments thereof tropic for a particular cell type,antibodies for proteins which undergo internalization in cycling,proteins that target intracellular localization and enhanceintracellular half-life. The technique of receptor-mediated endocytosisis described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432(1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414(1990).

Where appropriate, gene delivery agents such as, e.g., integrationsequences can also be employed. Numerous integration sequences are knownin the art (see, e.g., Nunes-Duby et al., Nucleic Acids Res. 26:391-406,1998; Sadwoski, J. Bacteriol., 165:341-357, 1986; Bestor, Cell,122(3):322-325, 2005; Plasterk et al., TIG 15:326-332, 1999; Kootstra etal., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). These includerecombinases and transposases. Examples include Cre (Sternberg andHamilton, J. Mol. Biol., 150:467-486, 1981), lambda (Nash, Nature, 247,543-545, 1974), Flp (Broach, et al., Cell, 29:227-234, 1982), R(Matsuzaki, et al., J. Bacteriology, 172:610-618, 1990), cpC31 (see,e.g., Groth et al., J. Mol. Biol. 335:667-678, 2004), sleeping beauty,transposases of the mariner family (Plasterk et al., supra), andcomponents for integrating viruses such as AAV, retroviruses, andantiviruses having components that provide for virus integration such asthe LTR sequences of retroviruses or lentivirus and the ITR sequences ofMV (Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). Inaddition, direct and targeted genetic integration strategies may be usedto insert nucleic acid sequences encoding the chimeric fusion proteinsincluding CRISPR/CAS9, zinc finger, TALEN, and meganuclease gene-editingtechnologies.

In one aspect, the invention provides expression vectors for theexpression of the chimeric proteins (and/or additional agents) that areviral vectors. Many viral vectors useful for gene therapy are known(see, e.g., Lundstrom, Trends Biotechnol., 21: 1 17, 122, 2003.Illustrative viral vectors include those selected from Antiviruses (LV),retroviruses (RV), adenoviruses (AV), adeno-associated viruses (AAV),and a viruses, though other viral vectors may also be used. For in vivouses, viral vectors that do not integrate into the host genome aresuitable for use, such as a viruses and adenoviruses. Illustrative typesof a viruses include Sindbis virus, Venezuelan equine encephalitis (VEE)virus, and Semliki Forest virus (SFV). For in vitro uses, viral vectorsthat integrate into the host genome are suitable, such as retroviruses,AAV, and Antiviruses. In one embodiment, the invention provides methodsof transducing a human cell in vivo, comprising contacting a solid tumorin vivo with a viral vector of the invention.

In embodiments, the present invention provides a host cell, comprisingthe expression vector comprising the chimeric protein described herein.

Expression vectors can be introduced into host cells for producing thepresent chimeric proteins. Cells may be cultured in vitro or geneticallyengineered, for example. Useful mammalian host cells include, withoutlimitation, cells derived from humans, monkeys, and rodents (see, forexample, Kriegler in “Gene Transfer and Expression: A LaboratoryManual,” 1990, New York, Freeman & Co.). These include monkey kidneycell lines transformed by SV40 (e.g., COS-7, ATCC CRL 1651); humanembryonic kidney lines (e.g., 293, 293-EBNA, or 293 cells subcloned forgrowth in suspension culture, Graham et al., J Gen Virol 1977, 36:59);baby hamster kidney cells (e.g., BHK, ATCC CCL 10); Chinese hamsterovary-cells-DHFR (e.g., CHO, Urlaub and Chasin, Proc Natl Acad Sci USA1980, 77:4216); DG44 CHO cells, CHO-K1 cells, mouse sertoli cells(Mather, Biol Reprod 1980, 23:243-251); mouse fibroblast cells (e.g.,NIH-3T3), monkey kidney cells (e.g., CV1 ATCC CCL 70); African greenmonkey kidney cells. (e.g., VERO-76, ATCC CRL-1587); human cervicalcarcinoma cells (e.g., HELA, ATCC CCL 2); canine kidney cells (e.g.,MDCK, ATCC CCL 34); buffalo rat liver cells (e.g., BRL 3A, ATCC CRL1442); human lung cells (e.g., W138, ATCC CCL 75); human liver cells(e.g., Hep G2, HB 8065); and mouse mammary tumor cells (e.g., MMT060562, ATCC CCL51). Illustrative cancer cell types for expressing thechimeric proteins described herein include mouse fibroblast cell line,NIH3T3, mouse Lewis lung carcinoma cell line, LLC, mouse mastocytomacell line, P815, mouse lymphoma cell line, EL4 and its ovalbumintransfectant, E.G7, mouse melanoma cell line, B16F10, mouse fibrosarcomacell line, MC57, and human small cell lung carcinoma cell lines, SCLC #2and SCLC #7.

Host cells can be obtained from normal or affected subjects, includinghealthy humans, cancer patients, and patients with an infectiousdisease, private laboratory deposits, public culture collections such asthe American Type Culture Collection, or from commercial suppliers.

Cells that can be used for production of the present chimeric proteinsin vitro, ex vivo, and/or in vivo include, without limitation,epithelial cells, endothelial cells, keratinocytes, fibroblasts, musclecells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes,monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,granulocytes; various stem or progenitor cells, in particularhematopoietic stem or progenitor cells (e.g., as obtained from bonemarrow), umbilical cord blood, peripheral blood, fetal liver, etc. Thechoice of cell type depends on the type of tumor or infectious diseasebeing treated or prevented, and can be determined by one of skill in theart.

Production and purification of Fc-containing macromolecules (such asmonoclonal antibodies) has become a standardized process, with minormodifications between products. For example, many Fc containingmacromolecules are produced by human embryonic kidney (HEK) cells (orvariants thereof) or Chinese Hamster Ovary (CHO) cells (or variantsthereof) or in some cases by bacterial or synthetic methods. Followingproduction, the Fc containing macromolecules that are secreted by HEK orCHO cells are purified through binding to Protein A columns andsubsequently ‘polished’ using various methods. Generally speaking,purified Fc containing macromolecules are stored in liquid form for someperiod of time, frozen for extended periods of time or in some caseslyophilized. In embodiments, production of the chimeric proteinscontemplated herein may have unique characteristics as compared totraditional Fc containing macromolecules. In certain examples, thechimeric proteins may be purified using specific chromatography resins,or using chromatography methods that do not depend upon Protein Acapture. In embodiments, the chimeric proteins may be purified in anoligomeric state, or in multiple oligomeric states, and enriched for aspecific oligomeric state using specific methods. Without being bound bytheory, these methods could include treatment with specific buffersincluding specified salt concentrations, pH and additive compositions.In other examples, such methods could include treatments that favor oneoligomeric state over another. The chimeric proteins obtained herein maybe additionally ‘polished’ using methods that are specified in the art.In embodiments, the chimeric proteins are highly stable and able totolerate a wide range of pH exposure (between pH 3-12), are able totolerate a large number of freeze/thaw stresses (greater than 3freeze/thaw cycles) and are able to tolerate extended incubation at hightemperatures (longer than 2 weeks at 40 degrees C.). In embodiments, thechimeric proteins are shown to remain intact, without evidence ofdegradation, deamidation, etc. under such stress conditions.

Subjects and/or Animals

In embodiments, the subject and/or animal is a mammal, e.g., a human,mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, ornon-human primate, such as a monkey, chimpanzee, or baboon. Inembodiments, the subject and/or animal is a non-mammal, such, forexample, a zebrafish. In embodiments, the subject and/or animal maycomprise fluorescently-tagged cells (with e.g. GFP). In embodiments, thesubject and/or animal is a transgenic animal comprising a fluorescentcell.

In embodiments, the subject and/or animal is a human. In embodiments,the human is a pediatric human. In embodiments, the human is an adulthuman. In embodiments, the human is a geriatric human. In embodiments,the human may be referred to as a patient.

In certain embodiments, the human has an age in a range of from about 0months to about 6 months old, from about 6 to about 12 months old, fromabout 6 to about 18 months old, from about 18 to about 36 months old,from about 1 to about 5 years old, from about 5 to about 10 years old,from about 10 to about 15 years old, from about 15 to about 20 yearsold, from about 20 to about 25 years old, from about 25 to about 30years old, from about 30 to about 35 years old, from about 35 to about40 years old, from about 40 to about 45 years old, from about 45 toabout 50 years old, from about 50 to about 55 years old, from about 55to about 60 years old, from about 60 to about 65 years old, from about65 to about 70 years old, from about 70 to about 75 years old, fromabout 75 to about 80 years old, from about 80 to about 85 years old,from about 85 to about 90 years old, from about 90 to about 95 years oldor from about 95 to about 100 years old.

In embodiments, the subject is a non-human animal, and therefore theinvention pertains to veterinary use. In a specific embodiment, thenon-human animal is a household pet. In another specific embodiment, thenon-human animal is a livestock animal.

Kits

The invention provides kits that can simplify the administration of anyagent described herein. An illustrative kit of the invention comprisesany composition described herein in unit dosage form. In one embodiment,the unit dosage form is a container, such as a pre-filled syringe, whichcan be sterile, containing any agent described herein and apharmaceutically acceptable carrier, diluent, excipient, or vehicle. Thekit can further comprise a label or printed instructions instructing theuse of any agent described herein. The kit may also include a lidspeculum, topical anesthetic, and a cleaning agent for theadministration location. The kit can also further comprise one or moreadditional agent described herein. In one embodiment, the kit comprisesa container containing an effective amount of a composition of theinvention and an effective amount of another composition, such thosedescribed herein.

Any aspect or embodiment described herein can be combined with any otheraspect or embodiment as disclosed herein

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1: Characterization of TIGIT-Fc-OX40L Chimeric Proteins

In this example, chimeric proteins comprising TIGIT and OX40L domainswere biochemically and functionally characterized.

Murine TIGIT-Fc-OX40L chimeric proteins and human TIGIT-Fc-OX40Lchimeric proteins were prepared; their schematics are shown at the topof FIG. 3A and FIG. 3B. The mTIGIT-Fc-OX40L and hTIGIT-Fc-OX40L chimericproteins were run on SDS-PAGE under reducing conditions, followingtreatment with N+O deglycosylating enzyme, and/or after boiling. TheWestern blots of the mTIGIT-Fc-OX40L and hTIGIT-Fc-OX40L chimericproteins indicated the presence of a dominant dimeric band in thenon-reduced lanes (FIG. 3A and FIG. 3B, lane 2 in each blot), which wasreduced to a glycosylated monomeric band in the presence of the reducingagent, β-mercaptoethanol (FIG. 3A and FIG. 3B, lane 3 each blot). Asshown in FIGS. 3A and 3B, lane 3 in each bot, the chimeric protein ranas a monomer in the presence of both a reducing agent(β-mercaptoethanol) and an endoglycosidase (PNGase).

The binding capabilities of the mTIGIT-Fc-OX40L chimeric proteins andhTIGIT-Fc-OX40L chimeric proteins were then assayed. For each chimericprotein ELISAs were performed under the following conditions:Heavy+Light Chain captured and detected with Fc-HRP (FIG. 4A and FIG.4B, top left), CD155/PVR-His captured and detected with IgG (FIG. 4A andFIG. 4B, top right), and OX40-His captured and detected with an antibodydirected to mOX40L (FIG. 4A and FIG. 4B, bottom left). ThemTIGIT-Fc-OX40L chimeric protein was OX40-Fc captured and detected witha recombinant CD155 (FIG. 4A, bottom right, “Dual ELISA”) and thehTIGIT-Fc-OX40L chimeric protein was OX40-Fc captured and detected witha recombinant CD155, CD112 or CD113 protein (FIG. 4A, bottom right,“Dual ELISA”). These data indicate that the mTIGIT-Fc-OX40L chimericprotein and the hTIGIT-Fc-OX40L chimeric proteins bind each of theirtargets and can bind both targets contemporaneously.

Additional analyses were carried out to determine whether themTIGIT-Fc-OX40L chimeric protein could bind its targets on the surfaceof living cells. A cell line was generated to overexpress human PVR(i.e., CHOK1/PVR); these cells are useful for detecting binding of aTIGIT-containing construct to cell membrane-associated PVR. See, FIG.5A. A cell line was generated to overexpress Nectin-2 (i.e.,CHOK1/Nectin2); these cells are useful for detecting binding of aTIGIT-containing construct to cell membrane-associated Nectin-2. See,FIG. 5B. A cell line was generated to overexpress Nectin-3 (i.e.,CHOK1/Nectin3); these cells are useful for detecting binding of aTIGIT-containing construct to cell membrane-associated Nectin-3. See,FIG. 5C. Another cell line was prepared that overexpresses mOX40 (i.e.,CHOK1-mOX40). Each of the cell lines were based on Chinese hamster ovaryK1 (CHOK1).

mTIGIT-Fc-OX40L chimeric protein or mPD-1-Fc-OX40L chimeric protein wasincubated with the parental and over-expressing cell lines for 2 hours.Cells were collected, washed, and stained with antibodies for thedetection of the chimeric protein binding by flow cytometry. As shown inFIG. 6 (bottom left and right), and as expected, the chimeric proteinsdid not bind the parental cell line and at any concentration of chimericprotein. However, the mTIGIT-Fc-OX40L bound the CHOK1/PVR engineeredcell line in a concentration-dependent manner; based on this data anEC₅₀ value of 4.634 nM was calculated. See, FIG. 6 (top left). Incontrast, the mPD-1-Fc-OX40L bound to neither engineered cell line. Aswith the CHOK1/PVR engineered cell line, the mTIGIT-Fc-OX40L chimericprotein also bound the CHOK1-mOX40 engineered cell line in aconcentration-dependent manner; based on this data an EC₅₀ value of 1.7nM was calculated. See, FIG. 6 (right). These data indicate that thedifferent components of the chimeric protein are each capable of bindingits respective receptor/ligands on living cells.

Next a microarray screen was conducted to identify further bindingpartners of the hTIGIT-Fc-OX40L. FIG. 7 is a table of results showingthe identified binding partners of the hTIGIT-Fc-OX40L from a microarray(containing about 6,000 human membrane proteins). Each expected bindingpartner for hTIGIT-Fc-OX40L was identified by the screen. There was noevidence of non-specific binding to other human proteins, and binding toGalectin-1 is seen in the screen for all Fc-containing fusion proteins.

Example 2: Characterization of CD172a(SIRPα)-Fc-LIGHT Chimeric Proteins

In this example, chimeric proteins comprising LIGHT and CD172a(SIRPα)domains were biochemically and functionally characterized.

Murine CD172a(SIRPα)-Fc-LIGHT chimeric proteins and humanCD172a(SIRPα)-Fc-LIGHT chimeric proteins were prepared; their schematicsare shown at the top of FIG. 8A and FIG. 8B. The mCD172a(SIRPα)-Fc-LIGHTand hCD172a(SIRPα)-Fc-LIGHT chimeric proteins were run on SDS-PAGE underreducing conditions, following treatment with N+O deglycosylatingenzyme, and/or after boiling. The Western blots of themCD172a(SIRPα)-Fc-LIGHT and hCD172a(SIRPα)-Fc-LIGHT chimeric proteinsindicated the presence of a dominant dimeric band in the non-reducedlanes (FIG. 8A and FIG. 8B, lane 2 in each blot), which was reduced to aglycosylated monomeric band in the presence of the reducing agent,3-mercaptoethanol (FIG. 8A and FIG. 8B, lane 3 each blot). As shown inFIGS. 8A and 8B, lane 3 in each bot, the chimeric protein ran as amonomer in the presence of both a reducing agent (3-mercaptoethanol) andan endoglycosidase (PNGase).

The binding capabilities of the mCD172a(SIRPα)-Fc-LIGHT chimericproteins and hCD172a(SIRPα)-Fc-LIGHT chimeric proteins were thenassayed. For each chimeric protein ELISAs were performed under thefollowing conditions: Heavy+Light Chain captured and detected withFc-HRP (FIG. 9A and FIG. 9B, top left), CD47-His captured and detectedwith IgG (FIG. 9A and FIG. 9B, top right), a murine LTBR-His capturedand detected with an antibody directed to murine LIGHT (FIG. 9A, bottomleft) or human LTBR-His captured and detected with an antibody directedto human LIGHT (FIG. 9B, bottom left), and LTBR His+GST captured anddetected with an antibody directed to SIRPα (FIG. 9A and FIG. 9B, bottomright, “Dual ELISA”). These data indicate that themCD172a(SIRPα)-Fc-LIGHT chimeric protein and the hCD172a(SIRPα)-Fc-LIGHTchimeric proteins bind each of their targets and can bind both targetscontemporaneously.

Additional analyses were carried out to determine whether themCD172a(SIRPα)-Fc-LIGHT chimeric protein could bind its targets on thesurface of living cells. CHO-K1 cells expressing murine CD47 (FIG. 10A,left) or to CHO-K1 cells expressing murine LTbR (FIG. 10A, right) wereprepared.

mCD172a(SIRPα)-Fc-LIGHT chimeric protein was incubated with the parentaland over-expressing cell lines for 2 hours. Cells were collected,washed, and stained with antibodies for the detection of the chimericprotein binding by flow cytometry. As shown in FIG. 10A (left), and asexpected, the chimeric protein did not bind the parental cell line andat any concentration of chimeric protein. However, themCD172a(SIRPα)-Fc-LIGHT bound the mCD47 engineered cell line (FIG. 10A,left) and the mLTbR engineered cell line (FIG. 10A, right) inconcentration-dependent manners; based on this data an EC₅₀ value forCD47 of 18 nM and for mCD172a(SIRPα) of 24 nM were calculated.Additionally, hCD172a(SIRPα)-Fc-LIGHT chimeric protein was incubatedwith the mCD47 over-expressing cell line. The hCD172a(SIRPα)-Fc-LIGHTbound the mCD47 engineered cell line (FIG. 10B) inconcentration-dependent manner; based on this data an EC₅₀ value of 57nM was calculated. These data indicate that the different components ofthe chimeric protein are each capable of binding its respectivereceptor/ligands on living cells.

The inability of the human TIGIT-Fc-OX40L chimeric protein to bind tored blood cells and, thereby, causing hemolysis was then tested. Here,hCD172a(SIRPα)-Fc-LIGHT, hCD172a(SIRPα)-Fc-CD40, or CD47 specificantibodies (clone CC2C6 or CC900002) were contacted with cynomolgusmacaque red blood cells (RBCs; FIG. 11A, left top and bottom) or humanRBCs (FIG. 11B and FIG. 11C, all panels). When compared to treatmentswith Triton-X (as a positive control), neither hCD172a(SIRPα)-Fc-LIGHTnor hCD172a(SIRPα)-Fc-CD40 caused significant lysis of cynomolgusmacaque RBCs (FIG. 11A, bottom left); an example plate is shown (FIG.11A, right). Neither hCD172a(SIRPα)-Fc-LIGHT nor hCD172a(SIRPα)-Fc-CD40significantly bound to human RBCs (FIG. 11B, all panels). When comparedto treatments with Triton-X (as a positive control),hCD172a(SIRPα)-Fc-LIGHT or hCD172a(SIRPα)-Fc-CD40 caused nearlyundetectable levels of lysis of human RBCs (FIG. 11C, all panels); anexample plate is shown (FIG. 11C, right). These data indicate thathCD172a(SIRPα)-Fc-LIGHT and hCD172a(SIRPα)-Fc-CD40 do not causehemolysis of human RBCs as an unwanted side effect.

Example 3: Characterization of PD-1-Fc-LIGHT Chimeric Proteins

In this example, chimeric proteins comprising LIGHT and PD-1 domainswere biochemically and functionally characterized.

Murine PD-1-Fc-LIGHT chimeric proteins and human PD-1-Fc-LIGHT chimericproteins were prepared; their schematics are shown at the top of FIG.12A and FIG. 12B. The mPD-1-Fc-LIGHT and hPD-1-Fc-LIGHT chimericproteins were run on SDS-PAGE under reducing conditions, followingtreatment with N+O deglycosylating enzyme, and/or after boiling. TheWestern blots of the mPD-1-Fc-LIGHT and hPD-1-Fc-LIGHT chimeric proteinsindicated the presence of a dominant dimeric band in the non-reducedlanes (FIG. 12A and FIG. 12B, lane 2 in each blot), which was reduced toa glycosylated monomeric band in the presence of the reducing agent,3-mercaptoethanol (FIG. 12A and FIG. 12B, lane 3 each blot). As shown inFIGS. 12A and 12B, lane 3 in each bot, the chimeric protein ran as amonomer in the presence of both a reducing agent (3-mercaptoethanol) andan endoglycosidase (PNGase).

The binding capabilities of the mPD-1-Fc-LIGHT chimeric proteins andhPD-1-Fc-LIGHT chimeric proteins were then assayed. For each chimericprotein ELISAs were performed under the following conditions:Heavy+Light Chain captured and detected with Fc-HRP (FIG. 13A and FIG.13B, top left), a murine LTBR-His captured and detected with an antibodydirected to murine LIGHT (FIG. 13A, top right) or human LTBR-Hiscaptured and detected with biotinylated human LIGHT (FIG. 13B, topright), and mPD-L1 captured and detected with an antibody directed tomLIGHT (FIG. 13A, bottom left; “Dual ELISA”) or hPDL1-Fc captured anddetected with hLTBR-His/6×His-HRP (FIG. 13A, bottom left; “Dual ELISA”).These data indicate that the mPD-1-Fc-LIGHT chimeric protein and thehPD-1-Fc-LIGHT chimeric proteins bind each of their targets and can bindboth targets contemporaneously.

Additional analyses were carried out to determine whether the murinePD-1-Fc-LIGHT chimeric protein (FIG. 14A) or human PD-1-Fc-LIGHTchimeric protein (FIG. 14A) can bind its targets on the surface ofliving cells. CHO-K1 cells expressing murine mPD-L1 (FIG. 14A, left),CHO-K1 cells expressing murine LTbR (FIG. 14A, right) and CHO-K1 cellsexpressing human mPD-L1 (FIG. 14B, left), were prepared. ThemPD-1-Fc-LIGHT chimeric protein was incubated with the parental andover-expressing cell lines for 2 hours. Cells were collected, washed,and stained with antibodies for the detection of the chimeric proteinbinding by flow cytometry. As shown in FIG. 14A (left and right), and asexpected, the mPD-1-Fc-LIGHT chimeric protein did not bind the parentalcell lines and at any concentration of chimeric protein. However, themPD-1-Fc-LIGHT bound the mPD-L1 engineered cell line (FIG. 14A, left)and the mLTbR engineered cell line (FIG. 14A, right) inconcentration-dependent manners. Additionally, hPD-1-Fc-LIGHT chimericprotein was incubated with the hPD-L1 over-expressing cell line. ThehPD-1-Fc-LIGHT bound the hPD-L1 engineered cell line (FIG. 14B) in aconcentration-dependent manner; based on this data an EC₅₀ value of 48nM was calculated. These data indicate that the different components ofthe chimeric protein are each capable of binding its respectivereceptor/ligands on living cells.

Example 4: Characterization of TIGIT-Fc-LIGHT Chimeric Proteins

In this example, chimeric proteins comprising TIGIT and LIGHT domainswere biochemically and functionally characterized.

Murine TIGIT-Fc-LIGHT chimeric proteins and human TIGIT-Fc-LIGHTchimeric proteins were prepared; their schematics are shown at the topof FIG. 15A and FIG. 15B. The mTIGIT-Fc-LIGHT and hTIGIT-Fc-LIGHTchimeric proteins were run on SDS-PAGE under reducing conditions,following treatment with N+O deglycosylating enzyme, and/or afterboiling. The Western blots of the mTIGIT-Fc-LIGHT and hTIGIT-Fc-LIGHTchimeric proteins indicated the presence of a dominant dimeric band inthe non-reduced lanes (FIG. 15A and FIG. 15B, lane 2 in each blot),which was reduced to a glycosylated monomeric band in the presence ofthe reducing agent, 3-mercaptoethanol (FIG. 15A and FIG. 15B, lane 3each blot). As shown in FIGS. 15A and 15B, lane 3 in each bot, thechimeric protein ran as a monomer in the presence of both a reducingagent (3-mercaptoethanol) and an endoglycosidase (PNGase).

The binding capabilities of the mTIGIT-1-Fc-LIGHT chimeric proteins andhTIGIT-1-Fc-LIGHT chimeric proteins were then assayed. For each chimericprotein ELISAs were performed under the following conditions:Heavy+Light Chain captured and detected with Fc-HRP (FIG. 16A and FIG.16B, top left), CD155/PVR captured and detected with Fc-HRP (FIG. 16A,top right) or CD155-His captured and detected with (FIG. 16B, topright), and mLTBR-His captured and detected with an antibody directed tomLIGHT (FIG. 16A, bottom left) or hCD155-Fc captured and detected withhLTBR-His/6×His-HRP (FIG. 16B, bottom left; “Dual ELISA”). These dataindicate that the mTIGIT-1-Fc-LIGHT chimeric protein and thehTIGIT-1-Fc-LIGHT chimeric proteins bind each of their targets and, atleast the hTIGIT-1-Fc-LIGHT can bind both targets contemporaneously.

Additional analyses were carried out to determine whether the murineTIGIT-1-Fc-LIGHT chimeric protein (FIG. 17) can bind its targets on thesurface of living cells.

mTIGIT-1-Fc-LIGHT chimeric protein or mPD-1-Fc-LIGHT chimeric proteinwas incubated CHO-K1 cells expressing murine PVR or expressing murineNectin-2 (FIG. 17, left top), CHO-K1 cells expressing murine LTbR (FIG.17A, right), or parental CHO-K1 cells (FIG. 17, right and left bottom).

As shown in FIG. 17 (bottom left), and as expected, neither themTIGIT-1-Fc-LIGHT chimeric protein nor the mPD-1-Fc-LIGHT chimericprotein bound the parental cell lines, except at the highestconcentration of chimeric protein. However, the mTIGIT-1-Fc-LIGHT boundthe mPVR engineered cell line in concentration-dependent manner; basedon this data an EC₅₀ value of 214.9 nM was calculated (FIG. 17, lefttop). The mTIGIT-1-Fc-LIGHT also bound the mLTbR engineered cell line inconcentration-dependent manner; based on this data an EC₅₀ value of 11nM was calculated (FIG. 17, right). These data indicate that thedifferent components of the chimeric protein are each capable of bindingits respective receptor/ligands on living cells.

Example 5: Further Characterization of Chimeric Proteins

In this example, further experiments were performed with the humanTIGIT-Fc-LIGHT, human CD172a(SIRPα)-Fc-LIGHT, human PD-1-Fc-LIGHT,murine PD-1-Fc-LIGHT, human TIGIT-Fc-OX40L, human TIGIT-Fc-LIGHT, humanCD172a(SIRPα)-Fc-LIGHT chimeric proteins.

Binding affinity measurements for human TIGIT-Fc-LIGHT,CD172a(SIRPα)-Fc-LIGHT, or PD-1-Fc-LIGHT to human LTbR was collected bybiolayer interferometry using an Octet system (see, FIG. 18A, allpanels). Binding affinity measurements for human PD-1-Fc-LIGHT torecombinant human PD-L1 or PD-L2 across a range of concentrations wascollected by biolayer interferometry using an Octet system (see, FIG.18B).

Binding affinity measurements of human TIGIT-Fc-OX40L and TIGIT-Fc-LIGHTto recombinant human CD155/PVR (as compared to a one-sided TIGIT-Fcfusion protein control) was collected by biolayer interferometry usingan Octet system (see, FIG. 19A) Binding affinity measurements of humanCD172a(SIRPα)-Fc-LIGHT to recombinant human CD47 (as compared to asingle-sided CD172a(SIRPα)-Fc control, or one of the two CD47 specificantibody controls) was collected by biolayer interferometry using anOctet system (see, FIG. 19B). Binding affinity measurements of humanPD-1-Fc-LIGHT to recombinant human PD-L1 (as compared to a single-sidedPD-1-Fc control or an anti-PD-L1 control: Atezolizumab) was collected bybiolayer interferometry using an Octet system (see, FIG. 19C). Bindingaffinity measurements of human CD172a(SIRPα)-Fc-LIGHT, TIGIT-Fc-LIGHT,or PD-1-Fc-LIGHT to recombinant human LTbR (as compared to asingle-sided LIGHT-Fc fusion protein control or an anti-LTbR antibody)was collected by biolayer interferometry using an Octet system (see,FIG. 19D).

A functional ability of the LIGHT domain-containing chimeric proteins orTIGIT domain-containing chimeric proteins was determined in asuperantigen cytokine release assay. Here, increasing concentrations ofstaphylococcus enterotoxin B (SEB) were used to activate humanperipheral blood leukocytes in the presence of various test agents. Thequantity of IL-2 (FIG. 20A) or TNFα (FIG. 20B) secreted into the culturesupernatant was monitored as a functional readout of the ability of testagents to either block suppressive signaling events or co-stimulateimmune activating signals.

FIG. 20A and FIG. 20B show superantigen cytokine release assays whichdemonstrate the effects of the various antibodies, murineTIGIT-Fc-OX40L, murine CD172a(SIRPα)-Fc-LIGHT, murine TIGIT-Fc-LIGHT, ormurine PD-1-Fc-LIGHT chimeric proteins on murine peripheral bloodleukocytes activated by SEB. FIG. 20C shows a compilation of the dataacross multiple superantigen (SEB) concentrations. As shown in FIG. 20Aand FIG. 20C, when 50 and 200 ng/ml SEB was administered, each of thethree concentrations (10 nM, 100 nM, and 250 nM) for themCD172a(SIRPα)-Fc-LIGHT and the mPD-1-Fc-LIGHT provided significantlygreater secretion of IL2 than all the antibody controls. As shown inFIG. 20B, when 50 and 200 ng/ml SEB was administered, at 100 nm or 250nm concentrations, the mPD-1-Fc-LIGHT provided significantly greatersecretion of TNFα than all the antibody controls; when 200 ng/ml SEB wasadministered, all three concentrations of mCD172a(SIRPα)-Fc-LIGHT,provided significantly greater secretion of TNFα than all the antibodycontrols.

Moreover, FIG. 21A to FIG. 21C show superantigen cytokine release assayswhich demonstrate the effects of the various antibodies, humanTIGIT-Fc-LIGHT (FIG. 21A), human TIGIT-Fc-OX40L (FIG. 21B), humanPD-1-Fc-LIGHT, and human CD172a(SIRPα)-Fc-LIGHT (FIG. 21C) chimericproteins on human peripheral blood leukocytes activated by SEB.

Accordingly, the above data demonstrates LIGHT domain-containingchimeric proteins and TIGIT domain-containing chimeric proteins asdescribed herein are capable of binding each of its three bindingpartners; they functionally activate primary human leukocytes cells invitro.

Example 6: Improved Tumor Killing and Increased Survival from Treatmentswith Chimeric Proteins

The in vivo anti-tumor activity of the murine TIGIT-Fc-OX40L, murineCD172a(SIRPα)-Fc-LIGHT, murine TIGIT-Fc-LIGHT, and murine PD-1-Fc-LIGHTchimeric protein was analyzed using the CT26 mouse colorectal tumormodel. In one set of experiments, Balb/c mice were inoculated with CT26tumor. When tumors reached a diameter of 4 to 5 mm, mice were treatedwith the mTIGIT-Fc-OX40L, mCD172a(SIRPα)-Fc-LIGHT, mTIGIT-Fc-LIGHT, andmPD-1-Fc-LIGHT chimeric protein or an anti-TIGIT, anti-CD47, anti-OX40,or anti-PD-1 antibody or a combination of anti-TIGIT and anti-OX40antibodies.

The tumor growth for each treatment group was assessed as shown in FIG.22A (left and right panels). Specifically, the untreated mice developedtumors quickly and treatment with an antibody or antibodies appeared toslightly delay the development of tumors. In contrast, treating micewith the mTIGIT-Fc-OX40L, mCD172a(SIRPα)-Fc-LIGHT, mTIGIT-Fc-LIGHT, ormPD-1-Fc-LIGHT chimeric protein significantly inhibited growth and/ordelayed the development of tumors.

The overall survival percentage of mice through forty days after tumorinoculation was also assessed. All of the untreated mice died withintwenty days after tumor inoculation and none of the antibody orantibodies treated mice had a 40-day survival percentage greater than25%; see FIG. 22B, left panel. Significantly, all the mice treated withthe mTIGIT-Fc-OX40L, mCD172a(SIRPα)-Fc-LIGHT, mTIGIT-Fc-LIGHT, ormPD-1-Fc-LIGHT chimeric protein survived past forty days after tumorinoculation; see FIG. 22B, right panel.

These data indicate that in vivo treatments with a TIGIT-Fc-OX40L,CD172a(SIRPα)-Fc-LIGHT, TIGIT-Fc-LIGHT, or PD-1-Fc-LIGHT chimericprotein inhibits tumor growth and improves survival.

Example 7: Characterization of the Contribution of an Fc Domain in aLinker to Functionality of Chimeric Proteins

In this example, the contribution of an Fc domain in a linker tofunctionality of chimeric proteins of the present invention was assayed.Here, a PD-1-Fc-OX40L was used as a model for Fc-containing chimericproteins. Thus, the data presented below is relevant to chimericproteins of the present invention.

In its native state, PD-1 exists as monomer whereas OX40Ls tend todimerize due to electrostatic interactions between the OX40L domains; Fcdomains associate with each other via disulfide bonds, e.g., via theircysteine residue(s). Together, several inter-molecular interactions maycontribute to the quaternary structure of PD-1-Fc-OX40L. There are, atleast, four potential configurations of PD-1-Fc-OX40L, with the chimericprotein existing as a monomer, a dimer, a trimer, or a hexamer. See,FIG. 23.

The existence of monomeric and dimeric configurations of the chimericprotein was tested by exposing chimeric proteins to reducing andnon-reducing conditions and then running the proteins on SDS-PAGE. Undernon-reducing conditions (Reduced: “−”), the chimeric protein migrated inSDS-PAGE at about 200 kDa. Here, Western blots were probed withantibodies directed against PD-1, Fc, or OX40L in, respectively, theleft, middle, and right blots shown in FIG. 24. Since, the predictedmonomeric molecular weight of the chimeric protein is 57.6 kDa, the 200kDa species was expected to be, at least a dimer. However, under reducedconditions (Reduced: “+”), which reduces disulfide bonds (e.g., betweenFc domains), the chimeric protein migrated in SDS-PAGE at about 100 kDa.Since the 100 kDa species was heavier than expected, it was predictedthat the extra mass was due to glycosylation. Finally, chimeric proteinswere treated with Peptide-N-Glycosidase F (PNGaseF “+”) and run onSDS-PAGE under reduced conditions. Under these conditions, the chimericprotein migrated at about 57.6 kDa. These data suggest that the chimericprotein is glycosylated and exists naturally, at least, as a dimer; withdimerization likely due to disulfide bonding between Fc domains e.g.,via their cysteine residue(s).

SDS-PAGE gel methods do not accurately predict the molecular weight forhighly charged and/or large molecular weight proteins. Thus, chimericproteins were next characterized using Size Exclusion Chromatography(SEC). Unlike SDS-PAGE, in which the negatively-charged SDS reducescharge-based interactions between peptides, SEC does not use detergentsor reducing agents. When the PD-1-Fc-OX40L chimeric protein was run onSEC, none of the peaks were around 200 kDa. This suggests, thatnatively, the chimeric protein does not exist as a dimer. Instead, apeak having a size greater than 670 kDa was detected. See, FIG. 25. Thisand the prior data suggests that the PD-1-Fc-OX40L chimeric proteinexists as a hexamer in its native state.

As shown above, when run on SDS-PAGE under non-reducing conditions orunder reducing conditions, SDS in the sample and/or running bufferconverts the hexameric PD-1-Fc-OX40L chimeric protein into a predominantdimer or monomer, respectively, in the absence and presence of areducing agent. See, FIG. 26 (left gel). When run on native PAGE, whichlacks SDS, and in the absence of a reducing agent, the chimeric proteinexists as a hexamer. However, when run on native PAGE and in thepresence of a reducing agent (which reduces disulfide bonds) thechimeric protein migrated heavier than expected; as shown in FIG. 26(right gel, lane 2), with the chimeric protein failed to substantiallymigrate out of the loading well. This data suggests that the chimericprotein oligomerized into a higher-order protein. Thus, in chimericproteins, disulfide bonding appears to be important for controllinghigher-order oligomerization.

To further confirm this, chimeric proteins lacking an Fc domain wereconstructed, e.g., “PD-1-No Fc-OX40L”. Such chimeric proteins will nothave the disulfide bonding which occurs between Fc domains in thechimeric proteins described previously. As shown in FIG. 27, whenchimeric proteins lacking Fc domains are run on native PAGE, none of theprotein substantially migrated out of its loading well; again,suggesting that the “No Fc” chimeric proteins have formed aconcatemer-like complex comprising numerous proteins. Thus, omission ofthe Fc domain in a chimeric protein leads to formation of proteinaggregates. These data indicate that disulfide bonding, e.g., between Fcdomains on different chimeric proteins, stabilizes the chimeric proteinsand ensures that they each exist as a hexamer and not as a higher-orderprotein/concatemer. In other words, the Fc domain surprisingly putsorder to chimeric protein complexes. Lanes 1 to 4 respectively include2.5 μg, of PD-1-No Fc-OX40L, 5 μg of PD-1-No Fc-OX40L, 2.5 μg of PD-1-NoFc-OX40L, and 5 μg of PD-1-No Fc-OX40L

Shown in FIG. 28 is a model summarizing the above data and showing how ahexamer and concatamers form from chimeric proteins of the presentinvention. The illustrative chimeric protein (PD-1-Fc-OX40L) naturallyforms into a hexamer (due to electrostatic interactions between theOX40L domains and dimerization by Fc domains). However, in the absenceof the controlling effects off disulfide bonding between Fc domains,under reduced conditions for the PD-1-Fc-OX40L protein and due to theabsence of Fc domains in the PD-1-No Fc-OX40L, these latter chimericproteins form concatamers.

Additionally, chimeric proteins were constructed in which the Fc domain(as described herein) was replaced with Ficolin (which lacks cysteineresidues necessary for disulfide bonding between chimeric proteins). Aswith the No Fc chimeric proteins and chimeric proteins comprising an Fcand run on native PAGE and in the presence of a reducing agent (both ofwhich formed aggregates that do not migrate into a gel) chimericproteins comprising Ficolin appear to also form higher-order latticeswhich did not migrate into a gel. These data reinforce the conclusionthat disulfide binding is important for proper folding and function ofchimeric proteins of the present invention.

Finally, chimeric proteins were prepared using coiled Fc domains(CCDFc). Very little purified protein was delivered under functionalevaluation.

Accordingly, including an Fc domain in a linker of a chimeric protein(which is capable of forming disulfide bonds between chimeric proteins),helps avoid formation of insoluble and, likely, non-functional proteinconcatamers and/or aggregates.

Example 8: Characterization of Different Joining Linker Sequences forthe Chimeric Proteins

Different unique joining linker sequences (17 linkers) were identifiedwith varying characteristics (length, solubility, charge andflexibility). Constructs were then synthesized incorporating each ofthose 17 joining linker sequences into the ‘linker 2’ position, wherethe configuration of chimeric protein:

ECD 1-Joining Linker 1-Fc-Joining Linker 2-ECD 2

The production levels for those 17 constructs were tested in CHO cells.The following table provides a summary for the different joining linkersequences, characteristics of those joining linkers, the productionlevel (by A280), and the binding values (EC₅₀) based on FACS analysis toPD-L1 or OX40. Some variations in production levels and activity betweencertain joining linker sequences were determined.

TABLE 2 Summary for optional joining linker sequences Protein CHO-PD-L1HeLa-OX40 Joining Linker 2 Protein Name conc. A280 EC50 (nM) EC50 (nM)Sequence Characteristics PD-1_IgG4_OX40L (1) 0.17 27   6 IEGRMD (SEQLinker ID NO: 52) PD-1_IgG4_OX40L (2) 0.12 23  67 SKYGPPCPPCP IgG4 Hinge(SEQ ID NO: 50) Region PD-1_IgG4_OX40L (3) 0.15 25 140 GGGSGGGS Flexible(SEQ ID NO: 55) PD-1_IgG4_OX40L (4) 0.11 36 125 GGGSGGGGSG FlexibleGG (SEQ ID NO: 56) PD-1_IgG4_OX40L (5) 0.22 25  41 EGKSSGSGSESFlexible + soluble KST (SEQ ID NO: 57) PD-1_IgG4_OX40L (6) 0.12 26 171GGSG (SEQ ID Flexible NO: 58) PD-1_IgG4_OX40L (7) 0.11 27 195 GGSGGGSGGGFlexible SG (SEQ ID NO: 59) PD-1_IgG4_OX40L (8) 0.21 20  48 EAAAKEAAAKERigid Alpha Helix AAAK (SEQ ID NO: 60) PD-1_IgG4_OX40L (9) 0.23 45  87EAAAREAAARE Rigid Alpha Helix AAAREAAAR (SEQ ID NO: 61)PD-1_IgG4_OX40L (10) 0.13 52  62 GGGGSGGGGS Flexible GGGGSAS(SEQ ID NO: 62) PD-1_IgG4_OX40L (11) 0.07 25 100 GGGVPRDCG Flexible(SEQ ID NO: 53) PD-1_IgG4_OX40L (12) 0.11 33  70 GGGGAGGGG Flexible(SEQ ID NO: 63) PD-1_IgG4_OX40L (13) 0.12 38  60 GS (SEQ ID NO:Highly flexible 64) PD-1_IgG4_OX40L (14) 0.18 25  70 GSGSGS (SEQHighly flexible ID NO: 65) PD-1_IgG4_OX40L (15) 0.19 24  67 GSGSGSGSGSHighly flexible (SEQ ID NO: 66) PD-1_IgG4_OX40L (16) 0.11 34  77 GGGGSASFlexible (SEQ ID NO: 67) PD-1_IgG4_OX40L (17) 0.19 32  44 APAPAPAPAPARigid PAPAPAPAP (SEQ ID NO: 68)

Characterization of PD-1-IgG4-OX40L chimeric proteins with differentjoining linker sequences (17 linkers) by Western blot analysis is shownin FIG. 29A to FIG. 29Q. Specifically, each individual domain of thefusion construct was probed using an anti-PD-1, anti-Fc, or anti-OX40Lantibody. Results showed similar performance across each chimericprotein suggesting that all of the candidate joining linker sequenceswere functional.

Additionally, each purified protein with different linker sequences wasalso characterized by binding to PD-L1 or OX40 in ELISA assays (FIG.30), as well as cell-based flow cytometry assays (FIG. 31A to FIG. 31P).

Example 9: Production of Additional TIGIT-Containing Chimeric ProteinsComprising Extracellular Domains of Other Type II Proteins

In this example, additional chimeric proteins of the present inventionare described. Such additional chimeric proteins will be made similar tohow the TIGIT-Fc-OX40L, TIGIT-Fc-CD40L, or TIGIT-Fc-LIGHT chimericproteins were made, e.g., as described above in the Detailed Descriptionand in the present application's priority document: U.S. 62/464,002.

These additional chimeric proteins will have the general formula: ECD1-Joining Linker 1-Fc Domain-Joining Linker 2-ECD 2, in which ECD 1 isthe extracellular domain of TIGIT and ECD 2 is the extracellular domainof a Type II protein, other than CD40L, OX40L, or LIGHT. Exemplary TypeII proteins include 4-1BBL, CD30L, FasL, GITRL, TL1A, and TRAIL. Thesechimeric proteins may lack one or both of the joining linkers. ExemplaryJoining Linker 1s, Fc Domains, and Joining Linker 2s are described abovein Table 1; modular linkers useful for forming chimeric proteins andcomprising specific Joining Linker 1s, Fc Domains, and Joining Linker 2sare shown in FIG. 32.

Alternately, the additional chimeric proteins will be fusion proteinshaving the general formula: N terminus-(a)-(b)-(c)-C terminus, in which(a) is TIGIT, (b) is a linker comprising at least a portion of a Fcdomain, and (c) is the extracellular domain of a Type II protein otherthan CD40L, OX40L, or LIGHT. Exemplary Type II proteins include 4-1BBL,CD30L, FasL, GITRL, TL1A, and TRAIL. Exemplary linkers are describedabove in Table 1; modular linkers useful for forming chimeric proteinsand comprising specific Joining Linker 1s, Fc Domains, and JoiningLinker 2s are shown in FIG. 32.

The amino acid sequence for 4-1BBL, CD30L, FasL, GITRL, TL1A, and TRAIL,respectively, comprises SEQ ID NO: 12, 26, 30, 15, 18, and 40. The aminoacid sequence for extracellular domain of 4-1BBL, CD30L, FasL, GITRL,TL1A, and TRAIL, respectively, comprises SEQ ID NO: 13, 27, 31, 16, 19,and 41. The amino acid sequence for TIGIT comprises SEQ ID NO: 9 and theextracellular domain of TIGIT comprises SEQ ID NO: 10. The chimericproteins may comprise a variant of the above-mentioned sequences, e.g.,at least about 95% identical to an above-mentioned sequence.

According, the present invention further includes the followingadditional chimeric proteins and methods using the additional chimericproteins (e.g., in treating a cancer and/or treating an inflammatorydisease): TIGIT-Fc-4-1BBL, TIGIT-Fc-CD30L, TIGIT-Fc-FasL,TIGIT-Fc-GITRL, TIGIT-Fc-TL1A, and TIGIT-Fc-TRAIL.

The additional chimeric proteins will be characterized as describedabove for TIGIT-Fc-OX40L, TIGIT-Fc-CD40L, or TIGIT-Fc-LIGHT in Examples1 to 8, albeit with reagents (e.g., binding partners, recombinant targetcells, and cancer cell/tumor types) that are specific to the additionalchimeric proteins rather than as needed for characterizingTIGIT-Fc-OX40L, TIGIT-Fc-CD40L, or TIGIT-Fc-LIGHT. Thus, usingTIGIT-Fc-4-1BBL and TIGIT-Fc-CD40L as examples, characterizations ofTIGIT-Fc-4-1BBL akin to Example 1 can be performed using anti-TIGIT,anti-Fc, and anti-4-1BBL antibodies rather than the anti-TIGIT, anti-Fc,and anti-CD40L antibodies needed for TIGIT-Fc-CD40L.

As with the TIGIT-Fc-OX40L, TIGIT-Fc-CD40L, or TIGIT-Fc-LIGHT chimericproteins, the additional chimeric proteins will be effective in treatinga cancer and/or treating an inflammatory disease by blocking TIGIT(which inhibits the transmission of an immune inhibitory signal) andenhancing, increasing, and/or stimulating the transmission of an immunestimulatory signal via activating the receptor/ligand of one of 4-1BBL,CD30L, FasL, GITRL, TL1A, and TRAIL. Moreover, the additional chimericproteins will be effective in treating a cancer and/or an inflammatorydisease yet without the toxicity resulting and undesirable side-effects,e.g., GI complications, from treatments comprising a plurality ofantibodies, e.g., a blocking antibody and an agonist antibody for thereceptor/ligand of one of 4-1BBL, CD30L, FasL, GITRL, and TRAIL.

Example 10: Production of Additional LIGHT-Containing Chimeric ProteinsComprising Extracellular Domains of Other Type I Proteins

In this example, additional chimeric proteins of the present inventionare described. Such additional chimeric proteins will be made similar tohow the CD172a(SIRPα)-Fc-LIGHT, TIGIT-Fc-LIGHT, or PD-1-Fc-LIGHTchimeric proteins were made, e.g., as described above in the DetailedDescription and in the present application's priority document: U.S.62/464,002.

These additional chimeric proteins will have the general formula: ECD1-Joining Linker 1-Fc Domain-Joining Linker 2-ECD 2, in which ECD 1 isthe extracellular domain of a Type I protein other than CD172a(SIRPα),TIGIT, or PD-1 and ECD 2 is the extracellular domain of a LIGHT.Exemplary Type I proteins include TIM3 and BTLA. These chimeric proteinsmay lack one or both of the joining linkers. Exemplary Joining Linker1s, Fc Domains, and Joining Linker 2s are described above in Table 1;modular linkers useful for forming chimeric proteins and comprisingspecific Joining Linker 1s, Fc Domains, and Joining Linker 2s are shownin FIG. 32.

Alternately, the additional chimeric proteins will be fusion proteinshaving the general formula: N terminus-(a)-(b)-(c)-C terminus, in which(a) is the extracellular domain of a Type I protein other thanCD172a(SIRPα), TIGIT, or PD-1, (b) is a linker comprising at least aportion of a Fc domain, and (c) is the extracellular domain of LIGHT.Exemplary Type I proteins include TIM3 and BTLA. Exemplary linkers aredescribed above in Table 1; modular linkers useful for forming chimericproteins and comprising specific Joining Linker 1s, Fc Domains, andJoining Linker 2s are shown in FIG. 32.

The amino acid sequence for TIM3 and BTLA respectively, comprises SEQ IDNO: 36 and 24. The amino acid sequence for extracellular domain of TIM3and BTLA, respectively, comprises SEQ ID NO: 37 and 25. The amino acidsequence for LIGHT comprises SEQ ID NO: 1 and the extracellular domainof LIGHT comprises SEQ ID NO: 2. The chimeric proteins may comprise avariant of the above-mentioned sequences, e.g., at least about 95%identical to an above-mentioned sequence.

According, the present invention further includes the followingadditional chimeric proteins and methods using the additional chimericproteins (e.g., in treating a cancer and/or treating an inflammatorydisease): TIM3-Fc-LIGHT and BTLA-Fc-LIGHT. The additional chimericproteins will be characterized as described above forCD172a(SIRPα)-Fc-LIGHT, TIGIT-Fc-LIGHT, or PD-1-Fc-LIGHT in Examples 1to 8, albeit with reagents (e.g., binding partners, recombinant targetcells, and cancer cell/tumor types) that are specific to the additionalchimeric proteins rather than as needed for characterizing TIM3-Fc-LIGHTand BTLA-Fc-LIGHT; as examples, characterizations of TIM3-Fc-LIGHT akinto Example 2 can be performed using anti-LIGHT, anti-Fc, and anti-TIM3antibodies rather than the anti-LIGHT, anti-Fc, and anti-CD172a(SIRPα)antibodies needed for CD172a(SIRPα)-Fc-LIGHT.

As with the CD172a(SIRPα)-Fc-LIGHT, TIGIT-Fc-LIGHT, or PD-1-Fc-LIGHTchimeric proteins, the additional chimeric proteins will be effective intreating a cancer and/or treating an inflammatory disease by blockingTIM3 and LIGHT (which inhibits the transmission of an immune inhibitorysignal) and enhancing, increasing, and/or stimulating the transmissionof an immune stimulatory signal via activating the receptor/ligand byLIGHT. Moreover, the additional chimeric proteins will be effective intreating a cancer and/or an inflammatory disease yet without thetoxicity resulting and undesirable side-effects, e.g., GI complications,from treatments comprising a plurality of antibodies, e.g., a blockingantibody and an agonist antibody for the receptor/ligand of one of TIM3and LIGHT.

EQUIVALENTS

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporatedby reference in their entireties.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

As used herein, all headings are simply for organization and are notintended to limit the disclosure in any manner. The content of anyindividual section may be equally applicable to all sections.

EQUIVALENTS

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

What is claimed is:
 1. A method for treating cancer comprisingadministering an effective amount of a pharmaceutical composition to asubject in need thereof, the pharmaceutical composition comprising achimeric protein of a general structure of:N terminus-(a)-(b)-(c)-C terminus, wherein: (a) is a first domaincomprising an extracellular domain of TIGIT, wherein said first domaincomprises an amino acid sequence that is at least 95% identical to theamino acid sequence of SEQ ID NO: 10, (b) is a linker comprising atleast one cysteine residue capable of forming a disulfide bond, and (c)is a second domain comprising an extracellular domain of LIGHT, whereinsaid second domain comprises an amino acid sequence that is at least 95%identical to the amino acid sequence of SEQ ID NO: 2, wherein the linkerconnects the first domain and the second domain.
 2. The method of claim1, wherein the first domain is capable of binding a TIGIT ligandselected from CD155/PVR, Nectin-2, Nectin-3, and Nectin-4.
 3. The methodof claim 1, wherein the second domain is capable of binding a LIGHTligand selected from LTBR, HVEM, and DcR3.
 4. The method of claim 1,wherein the linker is a polypeptide selected from a flexible amino acidsequence, an IgG hinge region, or an antibody sequence.
 5. The method ofclaim 1, wherein the linker comprises a hinge-CH2-CH3 Fc domain derivedfrom IgG1 or IgG4.
 6. The method of claim 5, wherein the hinge-CH2-CH3Fc domain is derived from human IgG1 or human IgG4.
 7. The method ofclaim 4, wherein the linker comprises an amino acid sequence that is atleast 95% identical to the amino acid sequence of SEQ ID NO: 46, SEQ IDNO: 47, or SEQ ID NO:
 48. 8. The method of claim 5, wherein the linkercomprises one or more joining linkers, such joining linkers beingindependently selected from SEQ ID NOs: 49-95, optionally: wherein thelinker comprises two or more joining linkers each joining linkerindependently selected from SEQ ID NOs: 49-95; wherein one joininglinker is N terminal to the hinge-CH2-CH3 Fc domain and another joininglinker is C terminal to the hinge-CH2-CH3 Fc domain.
 9. The method ofclaim 1, wherein: (i) the first domain comprises an amino acid sequencethat is at least 98% identical to the amino acid sequence of SEQ ID NO:10, and/or (ii) the second domain comprises an amino acid sequence thatis at least 98% identical to the amino acid sequence of SEQ ID NO: 2.10. The method of claim 1, wherein: (a) the first domain comprises theamino acid sequence of SEQ ID NO: 10, (b) the second domain comprisesthe amino acid sequence of SEQ ID NO: 2, and (c) the linker comprises anamino acid sequence that is at least 95% identical to the amino acidsequence of SEQ ID NO: 46, SEQ ID NO: 47, or SEQ ID NO:
 48. 11. Themethod of claim 1, wherein the chimeric protein has an amino acidsequence that is at least 95% identical to the amino acid sequence ofSEQ ID NO:
 11. 12. The method of claim 1, wherein the chimeric proteinis capable of forming a stable synapse between cells, optionally whereinthe stable synapse between cells provides spatial orientation thatfavors tumor reduction.
 13. The method of claim 12, wherein the spatialorientation positions T cells to attack tumor cells and/or stericallyprevents a tumor cell from delivering negative signals, includingnegative signals beyond those masked by the chimeric protein of theinvention.
 14. The method of claim 1, wherein binding of either or bothof the extracellular domains to its respective binding partner occurswith slow off rates (K_(off)), which provides a long interaction of areceptor and its ligand, optionally wherein the long interaction: (i)provides sustained negative signal masking effect; (ii) delivers alonger positive signal effect, optionally wherein the longer positivesignal effect allows an effector cell to be adequately stimulated for ananti-tumor effect; (iii) provides T cell proliferation and allows foranti-tumor attack; and/or (iv) allows sufficient signal transmission toprovide release of stimulatory signals, optionally wherein thestimulatory signal is a cytokine.
 15. The method of claim 1, wherein thesubject's T cells are activated.
 16. The method of claim 1, wherein thesubject has a tumor and one or more tumor cells are prevented fromtransmitting an immunosuppressive signal.
 17. The method of claim 1,wherein the subject's T cells are activated when bound by the seconddomain of the chimeric protein and: (a) one or more tumor cells areprevented from transmitting an immunosuppressive signal when bound bythe first domain of the chimeric protein, (b) a quantifiable cytokineresponse in the peripheral blood of the subject is achieved, and/or (c)tumor growth is reduced in the subject in need thereof as compared to asubject treated with antibodies directed to the TIGIT, LIGHT, or theirrespective ligands or receptors.
 18. The method of claim 1, wherein themethod reduces the amount or activity of regulatory T cells (Tregs) ascompared to untreated subjects or subjects treated with antibodiesdirected to the TIGIT, LIGHT, or their respective ligands or receptors.19. The method of claim 1, wherein the method increases priming ofeffector T cells in draining lymph nodes of the subject as compared tountreated subjects or subjects treated with antibodies directed to theTIGHT, LIGHT, or their respective ligands or receptors.
 20. The methodof claim 1, wherein the method causes an overall decrease inimmunosuppressive cells and a shift toward a more inflammatory tumorenvironment as compared to untreated subjects or subjects treated withantibodies directed to TIGIT or LIGHT, or their respective ligands orreceptors.